Proteases and methods for producing them

ABSTRACT

A secreted proteolytic polypeptide comprising at least three non-polar or uncharged polar amino acids within the last four amino acids of the C-terminus of the polypeptide, encoding polynucleotides, expression vectors comprising the polynucleotides, host cell comprising the polynucleotides, methods for producing said polypeptide, and methods for using the polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national applicationPCT/DK2004/000431, filed 21 Jun. 2004, which claims priority or thebenefit under 35 U.S.C. 119 of Danish applications Nos. PA 2003 00911filed Jun. 19, 2003, PA 2003 01846 filed Dec. 12, 2003, PA 2004 00335filed Mar. 1, 2004 and U.S. provisional applications Nos. 60/480,103filed Jun. 20, 2003, 60/531,073 filed Dec. 18, 2003, and 60/549,763filed Mar. 2, 2004 the contents of which are fully incorporated hereinby reference.

FIELD OF INVENTION

A number of microbially derived related proteases are notably difficultto produce in industrially relevant yields, they may be prone to varioustypes of degradation and/or instabilities. The present inventionprovides methods for producing such proteases by expressing them withC-terminal amino acid extensions and/or modifications of an existingC-terminus. The invention further provides the resulting proteasescomprising such amino acid extensions.

The present invention relates to isolated polypeptides having proteaseactivity related to a Nocardiopsis sp. protease, and isolated nucleicacid sequences encoding such proteases. The invention furthermorerelates to nucleic acid constructs, vectors, and host cells comprisingthese nucleic acid sequences as well as methods for producing and usingthe proteases, in particular within animal feed.

BACKGROUND

Polypeptides having protease activity, or proteases, are sometimes alsodesignated peptidases, proteinases, peptide hydrolases, or proteolyticenzymes. Proteases may be of the exo-type that hydrolyses peptidesstarting at either end thereof, or of the endo-type that act internallyin polypeptide chains (endopeptidases). Endopeptidases show activity onN— and C-terminally blocked peptide substrates that are relevant for thespecificity of the protease in question.

The term “protease” is defined herein as an enzyme that hydrolysespeptide bonds. It includes any enzyme belonging to the EC 3.4 enzymegroup (including each of the thirteen subclasses thereof). The EC numberrefers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, SanDiego, Calif., including supplements 1-5 published in Eur. J. Biochem.1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650; respectively.The nomenclature is regularly supplemented and updated; see e.g. theWorld Wide Web (WWW) at www.chem.qmw.ac.uk/iubmb/enzyme/index.html).

US patent publication No. 2002/0182672A1 discloses, that if one or twoof the last two amino acids at the C-terminus of a polypeptide is/arecharged polar D or E (negatively charged) or K, R, or H (positivelycharged), the tail would be considered polar, charged, and this makesthe polypeptide resistant against proteolytic degradation by a subclassof proteases that recognize non-polar C-terminal tails of secretedproteins.

Another disclosure reported, that proline residues at the C-terminus ofnascent polypeptide chains induce degradation of the polypeptide (2002.Prolin residues at the C terminus of nascent chains induce SsrA taggingduring translation termination. J. Biol. Chem. 277:33825-33823).

SUMMARY OF THE INVENTION

It is a well-known problem in the art of expressing polypeptides havingproteolytic activity, that many of such polypeptides are inherentlyunstable, they may be subject to autoproteolysis, or they may betargeted for degradation by other proteases already during theirproduction, resulting in sub-optimal yields. Many other factors maycontribute to their instability, not all of which are understood atpresent. It is of great interest to provide proteolytic polypeptideswith an increased stability, so that they may be produced in higheryields.

The present inventors provide herein proteolytic polypeptides of the S2Aand/or S1E protease classification, that comprise at least threenon-polar or uncharged polar amino acids within the last four aminoacids of the C-terminus of the polypeptide. The configuration of the atleast three non-polar or uncharged amino acid residues may be achievedby adding one or more amino acid(s) as a fusion-tail to the polypeptide,for instance by modifying the encoding polynucleotide to also encode theadditional amino acid(s). Another way could be to modify one or moreexisting C-terminal amino acid(s) in the polypeptide. These particularamino acid configurations at the C-terminus of the polypeptide of theinvention resulted in much improved yields as compared to the yields ofpolypeptides that did not have these C-terminal amino acidconfigurations, under otherwise identical conditions of production.

Accordingly, in a first aspect the invention relates to a secretedpolypeptide which has protease activity, preferably alpha-lyticendopeptidase activity, which polypeptide comprises at least threenon-polar or uncharged polar amino acids within the last four aminoacids of the C-terminus of the polypeptide, and which polypeptide:

-   -   (a) comprises an amino acid sequence which is at least 70%, or        preferably 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid        sequence of the mature part of the polypeptide shown in SEQ ID        NO: 28; SEQ ID NO: 33; SEQ ID NO: 37; SEQ ID NO: 41; SEQ ID NO:        43; or SEQ ID NO: 45;    -   (b) comprises an amino acid sequence which is at least 70%, or        preferably 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid        sequence of the the mature part of the polypeptide encoded by        the polynucleotide in SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 25;        SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 36; SEQ ID NO: 40; or        SEQ ID NO: 44;    -   (c) is encoded by a nucleic acid sequence which hybridizes under        very low, low, medium-low, medium, medium-high, high, or very        high stringency conditions with:        -   (I) a polynucleotide encoding the mature part of a protease,            said polynucleotide obtainable from genomic DNA from            Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235 by            use of primers SEQ ID NO's: 26 and 27; from Nocardiopsis            Alba DSM 15647 by use of primers SEQ ID NO's: 34 and 35;            from Nocardiopsis prasina DSM 15648 by use of primers SEQ ID            NO's: 38 and 39; or from Nocardiopsis prasina DSM 15649 by            use of primers SEQ ID NO's: 42 and 39;        -   (II) the polynucleotide of SEQ ID NO: 1; of SEQ ID NO: 2; of            SEQ ID NO: 25; of SEQ ID NO: 31; of SEQ ID NO: 32; of SEQ ID            NO: 36; of SEQ ID NO: 40; or of SEQ ID NO: 44;        -   (III) a subsequence of (I) or (II) of at least 500            nucleotides, preferably 400, 300, 200, or 100 nucleotides,            or        -   (IV) a complementary strand of (I), (II), or (III);    -   (d) comprises a mature part which is a variant of the mature        part of the polypeptide. having the amino acid sequence of SEQ        ID NO: 28; SEQ ID NO: 33; SEQ ID NO: 37; SEQ ID NO: 41; SEQ ID        NO: 43; or SEQ ID NO: 45, comprising a substitution, deletion,        extension, and/or insertion of one or more amino acids;    -   (e) is an allelic variant of (a), (b), (c), or (d); or    -   (f) is a fragment of (a), (b), (c), (d), or (e).

Preferably the polypeptide belongs to the S2A, or the S1E peptidasefamilies.

In a second aspect, the invention relates to an isolated polynucleotideencoding a polypeptide as defined in the first aspect.

Still, in a third aspect, the invention relates to a recombinantexpression vector or polynucleotide construct comprising apolynucleotide as defined in the previous aspect.

Yet a fourth aspect relates to a recombinant host cell comprising apolynucleotide as defined in the second aspect, or an expression vectoror polynucleotide construct as defined in the previous aspect.

In a fifth aspect, the invention also relates to a transgenic plant, orplant part, comprising a polynucleotide as defined in the second aspect,or an expression vector or polynucleotide construct as defined in thethird aspect.

The sixth aspect of the invention relates to a transgenic, non-humananimal, or products, or elements thereof, comprising a polynucleotide asdefined in the second aspect, or an expression vector or polynucleotideconstruct as defined in the third aspect.

The seventh aspect of the invention relates to a method for producing apolypeptide as defined in the first aspect, the method comprising: (a)cultivating a recombinant host cell as defined in the fourth aspect, ora transgenic plant or animal as defined in the fifth or sixth aspects,to produce a supernatant comprising the polypeptide, and optionally (b)recovering the polypeptide.

Other aspects of then invention relate to: an animal feed additivecomprising at least one polypeptide as defined in the first aspect; and

(a) at least one fat-soluble vitamin, and/or

(b) at least one water-soluble vitamin, and/or

(c) at least one trace mineral;

an animal feed composition having a crude protein content of 50 to 800g/kg and comprising at least one polypeptide as defined in the firstaspect, or at least one feed additive of the previous aspect;

a composition comprising at least one polypeptide as defined in thefirst aspect, together with at least one other enzyme selected fromamongst phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8);galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease(EC 3.4.-.-), phospholipase Al (EC 3.1.1.32); phospholipase A2 (EC3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3);phospholipase D (EC 3.1.4.4); and/or beta-glucanase (EC 3.2.1.4 or EC3.2.1.6);

a method for using at least one polypeptide as defined in the firstaspect, for improving the nutritional value of an animal feed, forincreasing digestible and/or soluble protein in animal diets, forincreasing the degree of hydrolysis of proteins in animal diets, and/orfor the treatment of vegetable proteins, the method comprising includingthe polypeptide(s) in animal feed, and/or in a composition for use inanimal feed;

a method for using at least one polypeptide as defined in the firstaspect, comprising including the polypeptide(s) in a detergentformulation.

DETAILED DESCRIPTION OF THE INVENTION

Proteases are classified on the basis of their catalytic mechanism intothe following groups: Serine proteases (S), Cysteine proteases (C),Aspartic proteases (A), Metalloproteases (M), and Unknown, or as yetunclassified, proteases (U), see Handbook of Proteolytic Enzymes, A. J.Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998), inparticular the general introduction part.

Serine proteases are ubiquitous, being found in viruses, bacteria andeukaryotes; they include exopeptidase, endopeptidase, oligopeptidase andomega-peptidase activity. Over 20 families (denoted S1-S27) of serineproteases have been identified, these being grouped into 6 clans denotedSA, SB, SC, SE, SF, and SG, on the basis of structural similarity andfunctional evidence (Barrett et al. 1998. Handbook of proteolyticenzymes). Structures are known for at least four of the clans (SA, SB,SC and SE), these appear to be totally unrelated, suggesting at leastfour evolutionary origins of serine peptidases. Alpha-lyticendopeptidases belong to the chymotrypisin (SA) clan, within which theyhave been assigned to subfamily A of the S2 family (S2A).

Another classification system of proteolytic enzymes is based onsequence information, and is therefore used more often in the art ofmolecular biology; it is described in Rawlings, N. D. et al., 2002,MEROPS: The protease database. Nucleic Acids Res. 30:343-346. The MEROPSdatabase is freely available electronically at on the World Wide Web atwww.merops.ac.uk. According to the MEROPS system, the proteolyticenzymes classified as S2A in ‘The Handbook of Proteolytic Enzymes’, arein MEROPS classified as ‘S1E’ proteases (Rawlings N D, Barrett A J.(1993) Evolutionary families of peptidases, Biochem. J. 290:205-218).

In particular embodiments, the proteases of the invention and for useaccording to the invention are selected from the group consisting of:

-   (a) proteases belonging to the EC 3.4.-.- enzyme group;-   (b) Serine proteases belonging to the S group of the above Handbook;-   (c1) Serine proteases of peptidase family S2A;-   (c2) Serine proteases of peptidase family S1E as described in    Biochem. J. 290:205-218 (1993) and in MEROPS a protease database,    release 6.20, Mar. 24, 2003, (www.merops.ac.uk). The database is    described in Rawlings, N. D., O'Brien, E. A. & Barrett, A. J. (2002)    MEROPS: the protease database. Nucleic Acids Res. 30, 343-346.

For determining whether a given protease is a Serine protease, and afamily S2A protease, reference is made to the above Handbook and theprinciples indicated therein. Such determination can be carried out forall types of proteases, be it naturally occurring or wild-typeproteases; or genetically engineered or synthetic proteases.

Protease activity can be measured using any assay, in which a substrateis employed, that includes peptide bonds relevant for the specificity ofthe protease in question. Assay-pH and assay-temperature are likewise tobe adapted to the protease in question. Examples of assay-pH-values arepH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples of assay-temperaturesare 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 95° C.

Examples of protease substrates are casein, such as Azurine-CrosslinkedCasein (AZCL-casein). Two protease assays are described in Example 2herein, either of which can be used to determine protease activity. Forthe purposes of this invention, the so-called pNA Assay is a preferredassay.

There are no limitations on the origin of the protease of the inventionand/or for use according to the invention. Thus, the term proteaseincludes not only natural or wild-type proteases obtained frommicroorganisms of any genus, but also any mutants, variants, fragmentsetc. thereof exhibiting protease activity, as well as syntheticproteases, such as shuffled proteases, and consensus proteases. Suchgenetically engineered proteases can be prepared as is generally knownin the art, eg by Site-directed Mutagenesis, by PCR (using a PCRfragment containing the desired mutation as one of the primers in thePCR reactions), or by Random Mutagenesis. The preparation of consensusproteins is described in eg EP 897985. The term “obtained from” as usedherein in connection with a given source shall mean that the polypeptideencoded by the nucleic acid sequence is produced by the source or by acell in which the nucleic acid sequence from the source is present. In apreferred embodiment, the polypeptide is secreted extracellularly.

In a specific embodiment, the protease is a low-allergenic variant,designed to invoke a reduced immunological response when exposed toanimals, including man. The term immunological response is to beunderstood as any reaction by the immune system of an animal exposed tothe protease. One type of immunological response is an allergic responseleading to increased levels of IgE in the exposed animal. Low-allergenicvariants may be prepared using techniques known in the art. For examplethe protease may be conjugated with polymer moieties shielding portionsor epitopes of the protease involved in an immunological response.Conjugation with polymers may involve in vitro chemical coupling ofpolymer to the protease, e.g. as described in WO 96/17929, WO 98/30682,WO 98135026, and/or WO 99/00489. Conjugation may in addition oralternatively thereto involve in vivo coupling of polymers to theprotease. Such conjugation may be achieved by genetic engineering of thenucleotide sequence encoding the protease, inserting consensus sequencesencoding additional glycosylation sites in the protease and expressingthe protease in a host capable of glycosylating the protease, see e.g.WO 00/26354. Another way of providing low-allergenic variants is geneticengineering of the nucleotide sequence encoding the protease so as tocause the protease to self-oligomerize, effecting that protease monomersmay shield the epitopes of other protease monomers and thereby loweringthe antigenicity of the oligomers. Such products and their preparationis described e.g. in WO 96/16177. Epitopes involved in an immunologicalresponse may be identified by various methods such as the phage displaymethod described in WO 00/26230 and WO 01/83559, or the random approachdescribed in EP 561907. Once an epitope has been identified, its aminoacid sequence may be altered to produce altered immunological propertiesof the protease by known gene manipulation techniques such as sitedirected mutagenesis (see e.g. WO 00/26230, WO 00/26354 and/or WO00/22103) and/or conjugation of a polymer may be done in sufficientproximity to the epitope for the polymer to shield the epitope.

The first aspect of the invention relates to a secreted polypeptidewhich has protease activity, preferably alpha-lytic endopeptidaseactivity, which polypeptide comprises at least three non-polar oruncharged polar amino acids within the last four amino acids of theC-terminus of the polypeptide; and which polypeptide:

-   -   (a) comprises an amino acid sequence which is at least 70%, or        preferably 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid        sequence of the mature part of the polypeptide shown in SEQ ID        NO: 28; SEQ ID NO: 33; SEQ ID NO: 37; SEQ ID NO: 41; SEQ ID NO:        43; or SEQ ID NO: 45;    -   (b) comprises an amino acid sequence which is at least 70%, or        preferably 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid        sequence of the the mature part of the polypeptide encoded by        the polynucleotide in SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 25;        SEQ ID NO: 31; SEQ ID NO: 32; SEQ ID NO: 36; SEQ ID NO: 40; or        SEQ ID NO: 44;    -   (c) is encoded by a nucleic acid sequence which hybridizes under        very low, low, medium-low, medium, medium-high, high, or very        high stringency conditions with:        -   (I) a polynucleotide encoding the mature part of a protease,            said polynucleotide obtainable from genomic DNA from            Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235 by            use of primers SEQ ID NO's: 26 and 27; from Nocardiopsis            Alba DSM 15647 by use of primers SEQ ID NO's: 34 and 35;            from Nocardiopsis prasina DSM 15648 by use of primers SEQ ID            NO's: 38 and 39; or from Nocardiopsis prasina DSM 15649 by            use of primers SEQ ID NO's: 42 and 39;        -   (II) the polynucleotide of SEQ ID NO: 1; of SEQ ID NO: 2; of            SEQ ID NO: 25; of SEQ ID NO: 31; of SEQ ID NO: 32; of SEQ ID            NO: 36; of SEQ ID NO: 40; or of SEQ ID NO: 44;        -   (III) a subsequence of (I) or (II) of at least 500            nucleotides, preferably 400, 300, 200, or 100 nucleotides,            or        -   (IV) a complementary strand of (I), (II), or (III);    -   (d) comprises a mature part which is a variant of the mature        part of the polypeptide having the amino acid sequence of SEQ ID        NO: 28; SEQ ID NO: 33; SEQ ID NO: 37; SEQ ID NO: 41; SEQ ID NO:        43; or SEQ ID NO: 45, comprising a substitution, deletion,        extension, and/or insertion of one or more amino acids;    -   (e) is an allelic variant of (a), (b), (c), or (d); or    -   (f) is a fragment of (a), (b), (c), (d), or (e).

For the purposes of the present invention, the degree of identitybetween two amino acid sequences, as well as the degree of identitybetween two nucleotide sequences, is determined by the program “align”which is a Needleman-Wunsch alignment (i.e. a global alignment). Theprogram is used for alignment of polypeptide, as well as nucleotidesequences. The default scoring matrix BLOSUM50 is used for polypeptidealignments, and the default identity matrix is used for nucleotidealignments. The penalty for the first residue of a gap is −12 forpolypeptides and −16 for nucleotides. The penalties for further residuesof a gap are −2 for polypeptides, and −4 for nucleotide.

“Align” is part of the FASTA package version v20u6 (see W. R. Pearsonand D. J. Lipman (1988), “Improved Tools for Biological SequenceAnalysis”, PNAS 85:2444-2448, and W. R. Pearson (1990) “Rapid andSensitive Sequence Comparison with FASTP and FASTA,” Methods inEnzymology 183:63-98). FASTA protein alignments use the Smith-Watermanalgorithm with no limitation on gap size (see “Smith-Watermanalgorithm”, T. F. Smith and M. S. Waterman (1981) J. Mol. Biol.147:195-197).

The degree of identity between two amino acid sequences may also bedetermined by the Clustal method (Higgins, 1989, CABIOS 5: 151-153)using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.)with an identity table and the following multiple alignment parameters:Gap penalty of 10, and gap length penalty of 10. Pairwise alignmentparameters are Ktuple=1, gap penalty=3, windows=5, and diagonals=5. Thedegree of identity between two nucleotide sequences may be determinedusing the same algorithm and software package as described above withthe following settings: Gap penalty of 10, and gap length penalty of 10.Pairwise alignment parameters are Ktuple=3, gap penalty=3 andwindows=20.

A fragment of one of the encoding polynucleotide sequences of theinvention is a polynucleotide which encodes a polypeptide having one ormore amino acids deleted from the amino and/or carboxyl terminuscompared to the full-length amino acid sequence. In one embodiment afragment encodes at least 75 amino acid residues, or at least 100 aminoacid residues, or at least 125 amino acid residues, or at least 150amino acid residues, or at least 160 amino acid residues, or at least165 amino acid residues, or at least 170 amino acid residues, or atleast 175 amino acid residues.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

The present invention also relates to isolated polypeptides havingprotease activity and which are encoded by nucleic acid sequences whichhybridize under very low, or low, or low-medium, medium, medium-high,high, or very high stringency conditions with a nucleic acid probe whichhybridizes under the same conditions with (I) a polynucleotide encodinga protease obtainable from genomic DNA from Nocardiopsis dassonvilleisubsp. dassonvillei DSM 43235 by use of primers SEQ ID NO's: 26 and 27;from Nocardiopsis Alba DSM 15647 by use of primers SEQ ID NO's: 34 and35; from Nocardiopsis prasina DSM 15648 by use of primers SEQ ID NO's:38 and 39; or from Nocardiopsis prasina DSM 15649 by use of primers SEQID NO's: 42 and 39; (II) the polynucleotide of SEQ ID NO: 1; of SEQ IDNO: 2; of SEQ ID NO: 25; of SEQ ID NO: 31; of SEQ ID NO: 32; of SEQ IDNO: 36; of SEQ ID NO: 40; or of SEQ ID NO: 44; (III) a subsequence of(I) or (II) of at least 500 nucleotides, preferably 400, 300, 200, or100 nucleotides, or (IV) a complementary strand of (I), (II), or (Ill)(J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, ALaboratory Manual, 2nd edition, Cold Spring Harbor, N.Y.). In oneparticular embodiment the nucleic acid probe is selected from amongstthe nucleic acid sequences of (a), (b), or (c) above. A polynucleotidecorresponding to the mature peptide encoding part of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 36,SEQ ID NO: 40, or SEQ ID NO: 44 is a preferred probe.

The nucleic acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 25,SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, or SEQ IDNO: 44, or a subsequence thereof, as well as the amino acid sequences ofSEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO:43, or SEQ ID NO: 45, or a fragment thereof, and even a genomicpolynucleotide encoding a protease obtainable from genomic DNA fromNocardiopsis dassonvillei subsp. dassonvillei DSM 43235 by use ofprimers SEQ ID NO's: 26 and 27; from Nocardiopsis Alba DSM 15647 by useof primers SEQ ID NO's: 34 and 35; from Nocardiopsis prasina DSM 15648by use of primers SEQ ID NO's: 38 and 39; or from Nocardiopsis prasinaDSM 15649 by use of primers SEQ ID NO's: 42 and 39, or a subsequencethereof, may be used to design a nucleic acid probe to identify andclone DNA encoding polypeptides having protease activity from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic or CDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, preferably at least 25,and more preferably at least 35 nucleotides in length. Longer probes canalso be used. Both DNA and RNA probes can be used. The probes aretypically labeled for detecting the corresponding gene (for example,with ³²p, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA that hybridizes with the probes described aboveand which encodes a polypeptide having protease activity. Genomic orother DNA from such other organisms may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA which is homologous with SEQ ID NO: 1or a subsequence thereof, the carrier material is used in a Southernblot. For purposes of the present invention, hybridization indicatesthat the nucleic acid sequence hybridizes to a labeled nucleic acidprobe corresponding to the nucleic acid sequence shown in SEQ ID NO: 1,its complementary strand, or a subsequence thereof, under very low tovery high stringency conditions. Molecules to which the nucleic acidprobe hybridizes under these conditions are detected using X-ray film.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS, 20% formamide preferably at least at 45° C. (very lowstringency), more preferably at least at 50° C. (low stringency), morepreferably at least at 55° C. (medium stringency), more preferably atleast at 60° C. (medium-high stringency), even more preferably at leastat 65° C. (high stringency), and most preferably at least at 70° C.(very high stringency).

For short probes about 15 nucleotides to about 70 nucleotides in length,stringency conditions are defined as prehybridization, hybridization,and washing post-hybridization at 5° C. to 10° C. below the calculatedT_(m) using the calculation according to Bolton and McCarthy (1962,Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 MNaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP40, 1× Denhardt'ssolution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate,0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southernblotting procedures.

For short probes about 15 nucleotides to about 70 nucleotides in length,the carrier material is washed once in 6×SSC plus 0.1% SDS for 15minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C.below the calculated T_(m).

The present invention also relates to variants of the polypeptide of theinvention, comprising a substitution, deletion, and/or insertion of oneor more amino acids.

In a particular embodiment, amino acid changes are of a minor nature,that is conservative amino acid substitutions that do not significantlyaffect the folding and/or activity of the protein; small deletions,typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small peptide of up to about 20-25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter the specific activityare known in the art and are described, for example, by H. Neurath andR. L. Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly as well as these inreverse.

In a particular embodiment, the polypeptides of the invention and foruse according to the invention are acid-stable. For the presentpurposes, the term acid-stable means that the residual activity after 2hours of incubation at pH 3.0 and 37° C., is at least 50%, as comparedto the residual activity of a corresponding sample incubated for 2 hoursat pH 9.0 and 5° C. In a particular embodiment, the residual activity isat least 60%, 70%, 80% or at least 90%.

In particular embodiments, the polypeptide of the invention is i) abacterial protease; ii) a protease of the phylum Actinobacteria; iii) ofthe class Actinobacteria; iv) of the order Actinomycetales v) of thefamily Nocardiopsaceae; vi) of the genus Nocardiopsis; and/or a proteasederived from vii) Nocardiopsis species such as Nocardiopsis alba,Nocardiopsis antarctica, Nocardiopsis prasina, composta, exhalans,halophila, halotolerans, kunsanensis, listeri, lucentensis, metallicus,synnemataformans, trehalosi, tropica, umidischolae, xinjiangensis, orNocardiopsis dassonvillei, for example Nocardiopsis dassonvillei DSM43235.

The above taxonomy is according to the chapter The road map to theManual by G. M. Garrity & J. G. Holt in Bergey's Manual of SystematicBacteriology, 2001, second edition, volume 1, David R. Bone, Richard W.Castenholz.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL). E.g., Nocardiopsis dassonvillei subsp.dassonvillei DSM 43235 is publicly available from DSMZ (DeutscheSammlung von Mlkroorganismen und Zellkulturen GmbH, Braunschweig,Germany). The strain was also deposited at other depositary institutionsas follows: ATCC 23219, IMRU 1250, NCTC 10489.

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The nucleic acid sequence may then be derived by similarlyscreening a genomic or cDNA library of another microorganism. Once anucleic acid sequence encoding a polypeptide has been detected with theprobe(s), the sequence may be isolated or cloned by utilizing techniqueswhich are known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

As defined herein, an “isolated” polypeptide is a polypeptide which isessentially free of other polypeptides, e.g., at least about 20% pure,preferably at least about 40% pure, more preferably about 60% pure, evenmore preferably about 80% pure, most preferably about 90% pure, and evenmost preferably about 95% pure, as determined by SDS-PAGE.

Polypeptides encoded by nucleic acid sequences of the present inventionalso include fused polypeptides or cleavable fusion polypeptides inwhich another polypeptide is fused at the N-terminus or the C-terminusof the polypeptide or fragment thereof. A fused polypeptide is producedby fusing a nucleic acid sequence (or a portion thereof) encodinganother polypeptide to a nucleic acid sequence (or a portion thereof) ofthe present invention. Techniques for producing fusion polypeptides areknown in the art, e.g. PCR, or ligating the coding sequences encodingthe polypeptides so that they are in frame and that expression of thefused polypeptide is under control of the same promoter(s) andterminator.

In the present context, non-polar amino acids are G, A, V, L, I, M, P, For W; and uncharged polar amino acids are S, T, N, Q, Y, og C. The terms“non-polar” and “uncharged polar” when used to describe amino acids in apolypeptide are generally recognized in the art as characterizing theside-chain of the amino acid. Hence, for instance, the free carboxylicacid of the c-terminal amino acid in a polypeptide is not consideredwhen determining whether this amino acid is a non-polar or unchargedpolar amino acid.

A preferred embodiment releates to a polypeptide of the first aspectwhich mature part is a wildtype polypeptide; an artificial variant of awildtype polypeptide said variant having one or more amino-acid(s) addedto the C-terminus as compared to the wildtype and preferably the one ormore added amino acid(s) is (are) non-polar or uncharged and even morepreferably the one or more added amino acid(s) is one or more of Q, S,V, A, or P; a shuffled polypeptide; or a protein-engineered polypeptide.

Another preferred embodiment relates to a polypeptide of the firstaspect, wherein the one or more added amino acids are selected from thegroup consisting of: QSHVQSAP, (SEQ ID NO:3), QSAP, (SEQ ID NO:3), QP,TL, TT, QL, TP, LP, TI, IQ, QP, PI, LT, TQ, IT, QQ, and PQ.

The inventors determined, that the polypeptides of the present inventionwere produced in even greater yields when they were expressed as matureproteases fused to a heterologous pro-region, as shown in the examplesbelow.

Accordingly, a preferred embodiment relates to the polypeptide accordingto the first aspect which when expressed and before maturation comprisesa heterologous pro-region from a protease; preferably the pro-region isderived from an S2A or S1E protease, more preferably the pro-region isencoded by a polynucleotide which hybridizes under very low, low,medium-low, medium, medium-high, high, or very high stringencyconditions with a polynucleotide encoding the pro-region shown inposition −166 to −1 of SEQ ID NO: 28, in position 1-166 of SEQ ID NO:30, in position −167 to −1 of SEQ ID NO: 33, in position −165 to −1 ofSEQ ID NO: 37, in position −165 to −1 of SEQ ID NO: 41, in position −165to −1 of SEQ ID NO: 43, in position −165 to −1 of SEQ ID NO: 45, inposition 1-165 of SEQ ID NO: 46, in position 1-166 of SEQ ID NO: 47, inposition 1-166 of SEQ ID NO: 48, in position 1-166 of SEQ ID NO: 49, inposition 1-166 of SEQ ID NO: 50, in position 1-165 of SEQ ID NO: 51, inposition 1-1 66 of SEQ ID NO: 52, or in position 1-1 66 of SEQ ID NO:53; and most preferably it is at least 70% identical, or preferably 75%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99%, identical to the pro-region shown in position −166 to −1 ofSEQ ID NO: 28, in position 1-166 of SEQ ID NO: 30, in position −167 to−1 of SEQ ID NO: 33, in position −165 to −1 of SEQ ID NO: 37, inposition −165 to −1 of SEQ ID NO: 41, in position −165 to −1 of SEQ IDNO: 43, in position −165 to −1 of SEQ ID NO: 45, in position 1-165 ofSEQ ID NO: 46, in position 1-166 of SEQ ID NO: 47, in position 1-166 ofSEQ ID NO: 48, in position 1-166 of SEQ ID NO: 49, in position 1-166 ofSEQ ID NO: 50, in position 1-165 of SEQ ID NO: 51, in position 1-166 ofSEQ ID NO: 52, or in position 1-166 of SEQ ID NO: 53.

When the particular C-teminal amino acid configuration of thepolypeptide of the invention was combined with an heterologous secretionsignal peptide fused to the N-terminal part of the polypeptide of theinvention, a synergy was achieved and a greater yield resulted.

Accordingly, a preferred embodiment of the invention relates to thepolypeptide of the first aspect which when expressed comprises aheterologous secretion signal-peptide which is cleaved from thepolypeptide when the polypeptide is secreted, preferably theheterologous secretion signal peptide is derived from a heterologousprotease; preferably the heterologous secretion signal peptide comprisesan amino acid sequence having a sequence identity of at least 70%, orpreferably 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99%, with the amino acid sequence encoded bypolynucleotides 1-81 of SEQ ID NO: 2, or SEQ ID NO: 44.

Nucleic Acid Sequences

The present invention also relates to isolated nucleic acid sequencesthat encode a polypeptide of the present invention. Particular nucleicacid sequences of the invention are the polynucleotides of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:36, SEQ ID NO: 40, or SEQ ID NO: 44. Another particular nucleic acidsequence of the invention is the sequence, preferably the maturepolypeptide encoding region thereof, which is obtainable from genomicDNA from Nocardiopsis dassonvillei subspecies dassonvillei DSM 43235.The present invention also encompasses nucleic acid sequences whichencode a polypeptide having the amino acid sequence of amino acids shownin positions 1 to 188, or positions −165 to 188, of SEQ ID NO: 43, whichnucleic acid sequences differ from the corresponding parts of SEQ ID NO:1 by virtue of the degeneracy of the genetic code. The present inventionalso relates to subsequences of of the above polynucleotides whichencode polypeptide fragments that have protease activity.

A subsequence of a polynucleotide is a nucleic acid sequence from whichone or more nucleotides from the 5′ and/or 3′ end has been deleted.Preferably, a subsequence contains at least 225 nucleotides, morepreferably at least 300 nucleotides, even more preferably at least 375,450, 500, 531, 600, 700, 800, 900 or 1000 nucleotides. The presentinvention also relates to nucleotide sequences which have a degree ofidentity to the polynucleotide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:25, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, or SEQID NO: 44 of at least 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, or at least 99%.

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide are known in the art and include isolation from genomicDNA, preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used. The nucleic acidsequence may be cloned from a strain of Nocardiopsis or another orrelated organism and thus, for example, may be an allelic or speciesvariant of the polypeptide encoding region of the nucleic acid sequence.

The term “isolated nucleic acid sequence” as used herein refers to anucleic acid sequence which is essentially free of other nucleic acidsequences, e.g., at least about 20% pure, preferably at least about 40%pure, more preferably at least about 60% pure, even more preferably atleast about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

Modification of a nucleic acid sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., variantsthat differ in specific activity, thermostability, pH optimum,allergenicity, or the like. The variant sequence may be constructed onthe basis of the nucleic acid sequence presented as the polypeptideencoding part of the polynucleotides of the invention, e.g. asubsequence thereof, and/or by introduction of nucleotide substitutionswhich do not give rise to another amino acid sequence of the polypeptideencoded by the nucleic acid sequence, but which correspond to the codonusage of the host organism intended for production of the protease, orby introduction of nucleotide substitutions which may give rise to adifferent amino acid sequence. For a general description of nucleotidesubstitution, see, e.g., Ford et al., 1991, Protein Expression andPurification 2: 95-107. Low-allergenic polypeptides can e.g. be preparedas described above.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by the isolated nucleic acidsequence of the invention, and therefore preferably not subject tosubstitution, may be identified according to procedures known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(see, e.g., Cunningham and Wells, 1989, Science 244: 1081-1085). In thelatter technique, mutations are introduced at every positively chargedresidue in the molecule, and the resultant mutant molecules are testedfor protease activity to identify amino acid residues that are criticalto the activity of the molecule. Sites of substrate-protease interactioncan also be determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labelling (see, e.g., de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, Journal of MolecularBiology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The present invention also relates to isolated nucleic acid sequencesencoding a polypeptide of the present invention, which hybridize undervery low stringency conditions, preferably low stringency conditions,more preferably medium stringency conditions, more preferablymedium-high stringency conditions, even more preferably high stringencyconditions, and most preferably very high stringency conditions with anucleic acid probe which hybridizes under the same conditions with thenucleic acid sequence of the invention or its complementary strand; orallelic variants and subsequences thereof (Sambrook et al., 1989,supra), as defined herein.

The present invention also relates to isolated nucleic acid sequencesproduced by (a) hybridizing a DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i) apolynucleotide of the invention, (ii) a subsequence of (i), or (iii) acomplementary strand of (i), or (ii); and (b) isolating the nucleic acidsequence. The subsequence is preferably a sequence of at least 100nucleotides such as a sequence that encodes a polypeptide fragment whichhas protease activity.

The introduction of a mutation into the nucleic acid sequence toexchange one nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure that utilizes a supercoiled, doublestranded DNA vector with an insert of interest and two synthetic primerscontaining the desired mutation. The oligonucleotide primers, eachcomplementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with DpnI which isspecific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used. The invention alsorelates to an isolated polynucleotide encoding a polypeptide as definedin the first aspect.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga nucleic acid sequence of the present invention operably linked to oneor more control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences. Expression will be understood to include any stepinvolved in the production of the polypeptide including, but not limitedto, transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid combined and juxtaposed in a manner that would not otherwise existin nature. The term nucleic acid construct is synonymous with the termexpression cassette when the nucleic acid construct contains all thecontrol sequences required for expression of a coding sequence of thepresent invention. The term “coding sequence” is defined herein as anucleic acid sequence that directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by a ribosome binding site (prokaryotes) or by the ATG startcodon (eukaryotes) located just upstream of the open reading frame atthe 5′ end of the mRNA and a transcription terminator sequence locatedjust downstream of the open reading frame at the 3′ end of the mRNA. Acoding sequence can include, but is not limited to, DNA, cDNA, andrecombinant nucleic acid sequences.

An isolated nucleic acid sequence encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the nucleic acid sequenceprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifying nucleicacid sequences utilizing recombinant DNA methods are well known in theart.

The term “control sequences” is defined herein to include all componentsthat are necessary or advantageous for the expression of a polypeptideof the present invention. Each control sequence may be native or foreignto the nucleic acid sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide. The term “operably linked” is defined herein asa configuration in which a control sequence is appropriately placed at aposition relative to the coding sequence of the DNA sequence such thatthe control sequence directs the expression of a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence that is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences that mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

Preferred terminators for bacterial host cells, such as a Bacillus hostcell, are the terminators from Bacillus licheniformis alpha-amylase gene(amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM),or the Bacillus amyloliquefaciens alpha-amylase gene (amyQ).

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformis alpha-amylase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Further signal peptides are described by Simonenand Palva, 1993, Microbiological Reviews 57: 109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal, peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide at such sites.Alternatively, the nucleic acid sequence of the present invention may beexpressed by inserting the nucleic acid sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleic acid sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis. Suitable markers foryeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents thereof. Preferredfor use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

For integration into the host cell genome, the vector may rely on thenucleic acid sequence encoding the polypeptide or any other element ofthe vector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1permitting replication in Bacillus. Examples of origins of replicationfor use in a yeast host cell are the 2 micron origin of replication,ARS1, ARS4, the combination of ARS1 and CEN3, and the combination ofARS4 and CEN6. The origin of replication may be one having a mutationwhich makes it functioning temperature-sensitive in the host cell (see,e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA75: 1433).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

The protease may also be co-expressed together with at least one otherenzyme of interest for animal feed, such as phytase (EC 3.1.3.8 or3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89);alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4.-.-), phospholipaseA1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4);and/or beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).

The enzymes may be co-expressed from different vectors, from one vector,or using a mixture of both techniques. When using different vectors, thevectors may have different selectable markers, and different origins ofreplication. When using only one vector, the genes can be expressed fromone or more promoters. If cloned under the regulation of one promoter(di- or multi-cistronic), the order in which the genes are cloned mayaffect the expression levels of the proteins. The protease may also beexpressed as a fusion protein, i.e. that the gene encoding the proteasehas been fused in frame to the gene encoding another protein. Thisprotein may be another enzyme or a functional domain from anotherenzyme.

Accordingly, the invention also relates to a recombinant expressionvector or polynucleotide construct comprising a polynucleotide of theinvention.

Host Cells

The present invention also relates to recombinant host cells, comprisinga nucleic acid sequence of the invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising anucleic acid sequence of the present invention is introduced into a hostcell so that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source. The host cell maybe a unicellular microorganism, e.g., a prokaryote, or a non-unicellularmicroorganism, e.g., a eukaryote.

Useful unicellular cells are bacterial cells such as gram positivebacteria including, but not limited to, a Bacillus cell, or aStreptomyces cell, or cells of lactic acid bacteria; or gram negativebacteria such as E. coli and Pseudomonas sp. Lactic acid bacteriainclude, but are not limited to, species of the genera Lactococcus,Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, andEnterococcus. Useful unicellular cells are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans orStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred embodiment, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus orBacillus subtilis cell. In another preferred embodiment, the Bacilluscell is an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278). The host cell maybe a eukaryote, such as a non-human animal cell, an insect cell, a plantcell, or a fungal cell. In one particular embodiment, the host cell is afungal cell. “Fungi” as used herein includes the phyla Ascomycota,Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworthet al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK) as well as theOomycota (as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

In another particular embodiment, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9,1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

Examples of filamentous fungal host cells are cells of species of, butnot limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor,Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, orTrichoderma.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

The invention relates to a recombinant host cell comprising apolynucleotide of the invention, or an expression vector orpolynucleotide construct of the invention. In a preferred embodiment,the recombinant host cell is a Bacillus cell.

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleic acid sequenceencoding a polypeptide having protease activity of the present inventionso as to express and produce the polypeptide in recoverable quantities.The polypeptide may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

In a particular embodiment, the polypeptide is targeted to the endospermstorage vacuoles in seeds. This can be obtained by synthesizing it as aprecursor with a suitable signal peptide, see Horvath et al in PNAS,Feb. 15, 2000, vol. 97, no. 4, p. 1914-1919.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot) or engineered variants thereof. Examples of monocot plantsare grasses, such as meadow grass (blue grass, Poa), forage grass suchas festuca, lolium, temperate grass, such as Agrostis, and cereals,e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana. Low-phytate plants as describede.g. in U.S. Pat. Nos. 5,689,054 and 6,111,168 are examples ofengineered plants.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers. Also specific plant tissues, such as chloroplast, apoplast,mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be aplant part. Furthermore, any plant cell, whatever the tissue origin, isconsidered to be a plant part.

Also included within the scope of the present invention are the progenyof such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

Conveniently, the expression construct is a nucleic acid construct whichcomprises a nucleic acid sequence encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleic acid sequence in the plant or plant partof choice. Furthermore, the expression construct may comprise aselectable marker useful for identifying host cells into which theexpression construct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences are determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV promoter may be used (Francket al., 1980, Cell 21: 285-294). Organ-specific promoters may be, forexample, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588).

A promoter enhancer element may also be used to achieve higherexpression of the protease in the plant. For instance, the promoterenhancer element may be an intron which is placed between the promoterand the nucleotide sequence encoding a polypeptide of the presentinvention. For instance, Xu et al., 1993, supra disclose the use of thefirst intron of the rice actin 1 gene to enhance expression.

Still further, the codon usage may be optimized for the plant species inquestion to improve expression (see Horvath et al referred to above).

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38).However it can also be used for transforming monocots, although othertransformation methods are generally preferred for these plants.Presently, the method of choice for generating transgenic monocots isparticle bombardment (microscopic gold or tungsten particles coated withthe transforming DNA) of embryonic calli or developing embryos(Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994, CurrentOpinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated thereinthe expression construct are selected and regenerated into whole plantsaccording to methods well-known in the art.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleic acid sequenceencoding a polypeptide having protease activity of the present inventionunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide. The invention relates to a transgenic plant,or plant part, comprising a polynucleotide as defined in claim 8, or anexpression vector or polynucleotide construct of the invention.

Animals

The present invention also relates to a transgenic, non-human animal andproducts or elements thereof, examples of which are body fluids such asmilk and blood, organs, flesh, and animal cells. Techniques forexpressing proteins, e.g. in mammalian cells, are known in the art, seee.g. the handbook Protein Expression: A Practical Approach, Higgins andHames (eds), Oxford University Press (1999), and the three otherhandbooks in this series relating to Gene Transcription, RNA processing,and Post-translational Processing. Generally speaking, to prepare atransgenic animal, selected cells of a selected animal are transformedwith a nucleic acid sequence encoding a polypeptide having proteaseactivity of the present invention so as to express and produce thepolypeptide. The polypeptide may be recovered from the animal, e.g. fromthe milk of female animals, or the polypeptide may be expressed to thebenefit of the animal itself, e.g. to assist the animal's digestion.Examples of animals are mentioned below in the section headed AnimalFeed.

To produce a transgenic animal with a view to recovering the proteasefrom the milk of the animal, a gene encoding the protease may beinserted into the fertilized eggs of an animal in question, e.g. by useof a transgene expression vector which comprises a suitable milk proteinpromoter, and the gene encoding the protease. The transgene expressionvector is microinjected into fertilized eggs, and preferably permanentlyintegrated into the chromosome. Once the egg begins to grow and divide,the potential embryo is implanted into a surrogate mother, and animalscarrying the transgene are identified. The resulting animal can then bemultiplied by conventional breeding. The polypeptide may be purifiedfrom the animal's milk, see e.g. Meade, H. M. et al (1999): Expressionof recombinant proteins in the milk of transgenic animals, Geneexpression systems: Using nature for the art of expression. J. M.Fernandez and J. P. Hoeffler (eds.), Academic Press.

In the alternative, in order to produce a transgenic non-human animalthat carries in the genome of its somatic and/or germ cells a nucleicacid sequence including a heterologous transgene construct including atransgene encoding the protease, the transgene may be operably linked toa first regulatory sequence for salivary gland specific expression ofthe protease, as disclosed in WO 2000064247.

The invention relates to a transgenic, non-human animal, or products, orelements thereof, comprising a polynucleotide, or an expression vectoror polynucleotide construct of the invention.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell or a transgenic plant or animal under conditions conducive forproduction of the polypeptide in a supernatant; and optionally (b)recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of a product, or disappearance of asubstrate. For example, a protease assay may be used to determine theactivity of the polypeptide as described herein.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purifcation, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Compositions

In a still further aspect, the present invention relates to compositionscomprising a polypeptide of the present invention. The polypeptidecompositions may be prepared in accordance with methods known in the artand may be in the form of a liquid or a dry composition. For instance,the polypeptide composition may be in the form of a granulate or amicrogranulate. The polypeptide to be included in the composition may bestabilized in accordance with methods known in the art. Examples aregiven below of preferred uses of the polypeptides or polypeptidecompositions of the invention.

Animal Feed

The present invention is also directed to methods for using thepolypeptides of the invention in animal feed, as well as to feedcompositions and feed additives comprising the polypeptides of theinvention. The term animal includes all animals, including human beings.Examples of animals are non-ruminants, and ruminants, such as cows,sheep and horses. In a particular embodiment, the animal is anon-ruminant animal. Non-ruminant animals include mono-gastric animals,e.g. pigs or swine (including, but not limited to, piglets, growingpigs, and sows); poultry such as turkeys, ducks and chicken (includingbut not limited to broiler chicks, layers); young calves; and fish(including but not limited to salmon, trout, tilapia, catfish and carps;and crustaceans (including but not limited to shrimps and prawns)

The term feed or feed composition means any compound, preparation,mixture, or composition suitable for, or intended for intake by ananimal.

In the use according to the invention the protease can be fed to theanimal before, after, or simultaneously with the diet. The latter ispreferred.

In a particular embodiment, the protease, in the form in which it isadded to the feed, or when being included in a feed additive, iswell-defined. Well-defined means that the protease preparation is atleast 50% pure as determined by Size-exclusion chromatography (seeExample 12 of WO 01/58275). In other particular embodiments the proteasepreparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95%pure as determined by this method. A well-defined protease preparationis advantageous. For instance, it is much easier to dose correctly tothe feed a protease that is essentially free from interfering orcontaminating other proteases. The term dose correctly refers inparticular to the objective of obtaining consistent and constantresults, and the capability of optimising dosage based upon the desiredeffect.

For the use in animal feed, however, the protease need not be that pure;it may e.g. include other enzymes, in which case it could be termed aprotease preparation. The protease preparation can be (a) added directlyto the feed (or used directly in a treatment process of vegetableproteins), or (b) it can be used in the production of one or moreintermediate compositions such as feed additives or premixes that issubsequently added to the feed (or used in a treatment process). Thedegree of purity described above refers to the purity of the originalprotease preparation, whether used according to (a) or (b) above.

Protease preparations with purities of this order of magnitude are inparticular obtainable using recombinant methods of production, whereasthey are not so easily obtained and also subject to a much higherbatch-to-batch variation when the protease is produced by traditionalfermentation methods. Such protease preparation may of course be mixedwith other enzymes.

In a particular embodiment, the protease for use according to theinvention is capable of solubilising vegetable proteins. A suitableassay for determining solubilised protein is disclosed in Example 11.

The term vegetable proteins as used herein refers to any compound,composition, preparation or mixture that includes at least one proteinderived from or originating from a vegetable, including modifiedproteins and protein-derivatives. In particular embodiments, the proteincontent of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60%(w/w). Vegetable proteins may be derived from vegetable protein sources,such as legumes and cereals, for example materials from plants of thefamilies Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, andPoaceae, such as soy bean meal, lupin meal and rapeseed meal. In aparticular embodiment, the vegetable protein source is material from oneor more plants of the family Fabaceae, e.g. soybean, lupine, pea, orbean.

In another particular embodiment, the vegetable protein source ismaterial from one or more plants of the family Chenopodiaceae, e.g.beet, sugar beet, spinach or quinoa. Other examples of vegetable proteinsources are rapeseed, and cabbage. Soybean is a preferred vegetableprotein source. Other examples of vegetable protein sources are cerealssuch as barley, wheat, rye, oat, maize (corn), rice, and sorghum.

The treatment according to the invention of vegetable proteins with atleast one protease of the invention results in an increasedsolubilisation of vegetable proteins. The following are examples of %solubilised protein obtainable using the proteases of the invention in amonogastric in vitro model: At least 102%, 103%, 104%, 105%, 106%, or atleast 107%, relative to a blank. The percentage of solubilised proteinis determined using the monogastric in vitro model of Example 11. Theterm solubilisation of proteins basically means bringing protein(s) intosolution. Such solubilisation may be due to protease-mediated release ofprotein from other components of the usually complex naturalcompositions such as feed.

In a further particular embodiment, the protease for use according tothe invention is capable of increasing the amount of digestiblevegetable proteins. The following are examples of % digested ordigestible protein obtainable using the proteases of the invention in amonogastric in vitro model: At least 104%, 105%, 106%, 107%, 108%, 109%,or at least 110%, relative to a blank. The percentage of digested ordigestible protein is determined using the in vitro model of Example 11.

The following are examples of % digested or digestible proteinobtainable using the proteases of the invention in an aquaculture invitro model: At least 103%, 104%, 105%, 106%, 107%, 108%, 109% or atleast 110%, relative to a blank. The percentage of digested ordigestible protein is determined using the aquaculture in vitro model ofExample 12.

In a still further particular embodiment, the protease for use accordingto the invention is capable of increasing the Degree of Hydrolysis (DH)of vegetable proteins. The following are examples of Degree ofHydrolysis increase obtainable in a monogastric in vitro model: At least102%, 103%, 104%, 105%, 106%, or at least 107%, relative to a blank. TheDH is determined using the monogastric in vitro model of Example 11. Thefollowing are examples of Degree of Hydrolysis increase obtainable in anaquaculture in vitro model: At least 102%, 103%, 104%, 105%, 106%, or atleast 107%, relative to a blank. The DH is determined using theaquaculture in vitro model of Example 12.

In a particular embodiment of a (pre-) treatment process of theinvention, the protease(s) in question is affecting (or acting on, orexerting its solubilising influence on) the vegetable proteins orprotein sources. To achieve this, the vegetable protein or proteinsource is typically suspended in a solvent, e.g. an aqueous solvent suchas water, and the pH and temperature values are adjusted paying dueregard to the characteristics of the enzyme in question. For example,the treatment may take place at a pH-value at which the activity of theactual protease is at least at least 40%, 50%, 60%, 70%, 80% or at least90%. Likewise, for example, the treatment may take place at atemperature at which the activity of the actual protease is at least40%, 50%, 60%, 70%, 80% or at least 90%. The above percentage activityindications are relative to the maximum activities. The enzymaticreaction is continued until the desired result is achieved, followingwhich it may or may not be stopped by inactivating the enzyme, e.g. by aheat-treatment step.

In another particular embodiment of a treatment process of theinvention, the protease action is sustained, meaning e.g. that theprotease is added to the vegetable proteins or protein sources, but itssolubilising influence is so to speak not switched on until later whendesired, once suitable solubilising conditions are established, or onceany enzyme inhibitors are inactivated, or whatever other means couldhave been applied to postpone the action of the enzyme.

In one embodiment the treatment is a pre-treatment of animal feed orvegetable proteins for use in animal feed.

The term improving the nutritional value of an animal feed meansimproving the availability and/or digestibility of the proteins, therebyleading to increased protein extraction from the diet components, higherprotein yields, increased protein degradation and/or improved proteinutilisation. The nutritional value of the feed is therefore increased,and the animal performance such as growth rate and/or weight gain and/orfeed conversion ratio (i.e. the weight of ingested feed relative toweight gain) of the animal is/are improved.

In a particular embodiment the feed conversion ratio is increased by atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or at least 10%. In a furtherparticular embodiment the weight gain is increased by at least 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10% or at least 11%. These figures are relativeto control experiments with no protease addition.

The feed conversion ratio (FCR) and the weight gain may be calculated asdescribed in EEC (1986): Directive de la Commission du 9 avril 1986fixant la méthode de calcul de la valeur énérgetique des alimentscomposés destinés á la volaille. Journal Officiel des CommunautésEuropéennes, L130, 53-54.

The protease can be added to the feed in any form, be it as a relativelypure protease, or in admixture with other components intended foraddition to animal feed, i.e. in the form of animal feed additives, suchas the so-called pre-mixes for animal feed.

In a further aspect the present invention relates to compositions foruse in animal feed, such as animal feed, and animal feed additives, e.g.premixes.

Apart from the protease of the invention, the animal feed additives ofthe inventon contain at least one fat-soluble vitamin, and/or at leastone water soluble vitamin, and/or at least one trace mineral. The feedadditive may also contain at least one macro mineral.

Further, optional, feed-additive ingredients are colouring agents, aromacompounds, stabilisers, antimicrobial peptides, including antifungalpolypeptides, and/or at least one other enzyme selected from amongstphytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4.-.-),phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4);lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3); phospholipaseD (EC 3.1.4.4); and/or beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).

In a particular embodiment these other enzymes are well-defined (asdefined above for protease preparations).

Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A,Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin,Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000),Plectasins, and Statins, including the compounds and polypeptidesdisclosed in PCT/DK02/00781 and PCT/DK02/00812, as well as variants orfragments of the above that retain antimicrobial activity.

Examples of antifungal polypeptides (AFP's) are the Aspergillusgiganteus, and Aspergillus niger peptides, as well as variants andfragments thereof which retain antifungal activity, as disclosed in WO94/01459 and WO 02/090384.

Usally fat- and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed. A premixenriched with a protease of the invention, is an example of an animalfeed additive of the invention.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This isso in particular for premixes.

The following are non-exclusive lists of examples of these components:

Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E,and vitamin K, e.g. vitamin K3.

Examples of water-soluble vitamins are vitamin B12, biotin and choline,vitamin B1, vitamin B2, vitamin B6, niacin, folic acid andpanthothenate, e.g. Ca-D-panthothenate.

Examples of trace minerals are manganese, zinc, iron, copper, iodine,selenium, and cobalt.

Examples of macro minerals are calcium, phosphorus and sodium.

The nutritional requirements of these components (exemplified withpoultry and piglets/pigs) are listed in Table A of WO 01/58275.Nutritional requirement means that these components should be providedin the diet in the concentrations indicated.

In the alternative, the animal feed additive of the invention comprisesat least one of the individual components specified in Table A of WO01/58275. At least one means either of, one or more of, one, or two, orthree, or four and so forth up to all thirteen, or up to all fifteenindividual components. More specifically, this at least one individualcomponent is included in the additive of the invention in such an amountas to provide an in-feed-concentration within the range indicated incolumn four, or column five, or column six of Table A.

The present invention also relates to animal feed compositions. Animalfeed compositions or diets have a relatively high content of protein.Poultry and pig diets can be characterised as indicated in Table B of WO01/58275, columns 2-3. Fish diets can be characterised as indicated incolumn 4 of this Table B. Furthermore such fish diets usually have acrude fat content of 200-310 g/kg. WO 01/58275 corresponds to U.S. Ser.No. 09/779334 which is hereby incorporated by reference.

An animal feed composition according to the invention has a crudeprotein content of 50-800 g/kg, and furthermore comprises at least oneprotease as claimed herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolisable energy of 10-30 MJ/kg; and/or a content ofcalcium of 0.1-200 g/kg; and/or a content of available phosphorus of0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or acontent of methionine plus cysteine of 0.1-150 g/kg; and/or a content oflysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolisable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO01/58275 (R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25,i.e. Crude protein (g/kg)=N (g/kg)×6.25. The nitrogen content isdetermined by the Kjeldahl method (A.O.A.C., 1984, Official Methods ofAnalysis 14th ed., Association of Official Analytical Chemists,Washington D.C.).

Metabolisable energy can be calculated on the basis of the NRCpublication Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C., pp. 26, and the European Table of Energy Values forPoultry Feed-stuffs, Spelderholt centre for poultry research andextension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen& looijen bv, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one vegetable protein or protein source as definedabove.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-10% fish meal; and/or 0-20% whey.

Animal diets can e.g. be manufactured as mash feed (non pelleted) orpelleted feed. Typically, the milled feed-stuffs are mixed andsufficient amounts of essential vitamins and minerals are addedaccording to the specifications for the species in question. Enzymes canbe added as solid or liquid enzyme formulations. For example, a solidenzyme formulation is typically added before or during the mixing step;and a liquid enzyme preparation is typically added after the pelletingstep. The enzyme may also be incorporated in a feed additive or premix.

The final enzyme concentration in the diet is within the range of0.01-200 mg enzyme protein per kg diet, for example in the range of0.5-25 mg enzyme protein per kg animal diet.

The protease should of course be applied in an effective amount, i.e. inan amount adequate for improving solubilisation and/or improvingnutritional value of feed. It is at present contemplated that the enzymeis administered in one or more of the following amounts (dosage ranges):0.01-200; 0.01-100; 0.5-100; 1-50; 5-100; 10-100; 0.05-50; or0.10-10—all these ranges being in mg protease enzyme protein per kg feed(ppm).

For determining mg enzyme protein per kg feed, the protease is purifiedfrom the feed composition, and the specific activity of the purifiedprotease is determined using a relevant assay (see under proteaseactivity, substrates, and assays). The protease activity of the feedcomposition as such is also determined using the same assay, and on thebasis of these two determinations, the dosage in mg enzyme protein perkg feed is calculated.

The same principles apply for determining mg enzyme protein in feedadditives. Of course, if a sample is available of the protease used forpreparing the feed additive or the feed, the specific activity isdetermined from this sample (no need to purify the protease from thefeed composition or the additive).

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

Detergent Compositions

The protease of the invention may be added to and thus become acomponent of a detergent composition. The detergent composition of theinvention may for example be formulated as a hand or machine laundrydetergent composition including a laundry additive composition suitablefor pre-treatment of stained fabrics and a rinse added fabric softenercomposition, or be formulated as a detergent composition for use ingeneral household hard surface cleaning operations, or be formulated forhand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additivecomprising the protease of the invention. The detergent additive as wellas the detergent composition may comprise one or more other enzymes suchas another protease, such as alkaline proteases from Bacillus, a lipase,a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, amannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., alaccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e. pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Suitable lipases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Examples of usefullipases include lipases from Humicola (synonym Thermomyces), e.g. fromH. lanuginosa (T. lanuginosus) as described in EP 258068 and EP 305216or from H. insolens as described in WO 96/13580, a Pseudomonas lipase,e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia(EP 331376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp.strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO96/12012), a Bacillus lipase, e.g. from B. subtilis (Dartois et al.(1993), Biochemica et Biophysica Acta, 1131, 253-360), B.stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422). Otherexamples are lipase variants such as those described in WO 92/05249, WO94/01541, EP 407225, EP 260105, WO 95/35381, WO 96/00292, WO 95/30744,WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.Preferred commercially available lipase enzymes include Lipolase™ andLipolase Ultra™ (Novozymes A/S).

Suitable amylases (alpha- and/or beta-) include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Amylases include, for example, alpha-amylases obtained fromBacillus, e.g. a special strain of B. licheniformis, described in moredetail in GB 1,296,839. Examples of useful amylases are the variantsdescribed in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424,especially the variants with substitutions in one or more of thefollowing positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188,190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from GenencorInternational Inc.).

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178,5,776,757 and WO 89/09259. Especially suitable cellulases are thealkaline or neutral cellulases having colour care benefits. Examples ofsuch cellulases are cellulases described in EP 0 495257, EP 531372, WO96/11262, WO 96/29397, WO 98/08940. Other examples are cellulasevariants such as those described in WO 94/07998, EP 0 531 315, U.S. Pat.Nos. 5,457,046, 5,686,593, 5,763,254, WO 95/24471, WO 98/12307 and WO99/01544. Commercially available cellulases include Celluzyme™, andCarezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (GenencorInternational Inc.), and KAC-500(B)™ (Kao Corporation).

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g. from C. cinereus, and variants thereof as those describedin WO 93/24618, WO 95/10602, and WO 98/15257. Commercially availableperoxidases include Guardzyme™ (Novozymes).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e. a separate additive or a combined additive, canbe formulated e.g. as a granulate, a liquid, a slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65 % of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of e.g. the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in e.g. WO 92/19709and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g. fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions anyenzyme, in particular the enzyme of the invention, may be added in anamount corresponding to 0.01-100 mg of enzyme protein per liter of washliqour, preferably 0.05-5 mg of enzyme protein per liter of wash liqour,in particular 0.1-1 mg of enzyme protein per liter of wash liqour.

The enzyme of the invention may additionally be incorporated in thedetergent formulations disclosed in WO 97/07202.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

EXAMPLES

Materials and Methods

Strains:

Bacillus subtilis PL1801 (Diderichsen, B et al. 1990. Cloning of aldB,which encodes alpha-acetolactate decarboxylase, an exoenzyme fromBacillus brevis. J. Bacteriol., 172, 4315-4321)

Bacillus subtilis MB1053

Bacillus subtilis PL3598-37

Bacillus subtilis MB1510

Bacillus subtilis PL2306. This strain is the B. subtilis DN1885 withdisrupted apr and npr genes (Diderichsen, B., Wedsted, U., Hedegaard,L., Jensen, B. R., Sjøholm, C. (1990) Cloning of aldB, which encodesalpha-acetolactate decarboxylase, an exoenzyme from Bacillus brevis. J.Bacteriol., 172, 4315-4321) which is also disrupted in thetranscriptional unit of the known Bacillus subtilis cellulase gene,resulting in cellulase negative cells. The disruption was performedessentially as described in (Eds. A. L. Sonenshein, J. A. Hoch andRichard Losick (1993) Bacillus subtilis and other Gram-PositiveBacteria, American Society for microbiology, p. 618).

Procedure for Isolating Genomic DNA.

5 Harvest 1.5 ml culture and resuspend in 100 μl TEL. Leave at 37C for30 min.

Add 500 μl thiocynate buffer and leave at room temperature for 10 min.

Add 250 μl NH4Ac and leave at ice for 10 min.

Add 500 μl CIA and mix.

Transfer to a microcentrifuge and spin for 10 min. at full speed.

10 Transfer supernatant to a new Eppendorf tube and add 0.54 volume coldisopropanol. Mix thoroughly.

Spin and wash the DNA pellet with 70% EtOH.

Resuspend the genomic DNA in 100 μl TER.

TE: 10 mM Tris-HCl, pH 7.4

-   -   1 mM EDTA, pH 8.0

TEL: 50 mg/ml Lysozym in TE-buffer

Thiocyanate: 5M guanidium thiocyanate

-   -   100 mM EDTA    -   0.6% w/v N-laurylsarcosine, sodium salt.    -   60 g thiocyanate, 20 ml 0.5 M EDTA, pH 8.0, 20 ml H2O dissolves        at 65 C. Cool down to RT and add 0.6 g N-laurylsarcosine. Add        H2O to 100 ml and filter it through a 0.2μ sterile filter.

NH4Ac: 7.5 M CH3COONH4

TER: 1 μg/ml Rnase A in TE-buffer

CIA: Chloroform/isoamyl alcohol 24:1

Purification of PCR Bands and DNA Sequencing

PCR fragment can be purified using GFX™ PCR DNA and Gel Band™Purification Kit (Pharmacia Biotech) according to the manufacturer'sinstructions. The nucleotide sequences of the amplified PCR fragmentsare determined on an ABI PRISM™ 3700 DNA Analyzer (Perkin Elmer, USA)using 50-100 ng as template, the Taq deoxy-terminal cycle sequencing kit(Perkin Elmer, USA), fluorescent labeled terminators and 5 pmol of thesequencing primer of choice.

Media

-   TY: (As described in Ausubel, F. M. et al. (eds.) “Current protocols    in Molecular Biology”. John Wiley and Sons, 1995).-   LB agar: (As described in Ausubel, F. M. et al. (eds.) “Current    protocols in Molecular Biology”. John Wiley and Sons, 1995).-   LB-PG agar: is LB agar supplemented with 0.5% Glucose and 0.05 M    potassium phosphate, pH 7.0.    Proteolytic Activity

S2A protease activity is measured using the PNA assay withsuccinyl-alanine-alanine-proline-phenylalnine-paranitroanilide as asubstrate unless otherwise mention. The principle of the PNA assay isdescribed in Rothgeb, T. M., Goodlander, B. D., Garrison, P. H., andSmith, L. A., Journal of the American Oil Chemists' Society, Vol. 65 (5)pp. 806-810 (1988).

Gene Expression in Bacillus subtilis Host

All the expressed genes in the following examples are integrated byhomologous recombination on the Bacillus subtilis host cell genome. Thegenes are expressed under the control of a triple promoter system (asdescribed in WO 99/43835), consisting of the promoters from Bacilluslicheniformis alpha-amylase gene (amyL), Bacillus amyloliquefaciensalpha-amylase gene (amyQ), and the Bacillus thuringiensis cryIIIApromoter including stabilizing sequence. The gene coding forChloramphenicol acetyl-transferase was used as maker. (Described in eg.Diderichsen, B.; Poulsen, G. B.; Joergensen, S. T.; A useful cloningvector for Bacillus subtilis. Plasmid 30:312 (1993)).

Example 1 Construction of Synthetic 10R Tail-variant Genes with SavinaseSignal

A synthetic 10R gene (10RS) encoding a S2A protease denoted 10R fromNocardiopsis sp. NRRL 18262 having the amino acid sequence shown in SEQID NO: 43 (WO 01/58276) was constructed, which has the nucleotidesequence shown in SEQ ID NO: 1. This synthetic gene was fused by PCR inframe to the DNA coding for the signal peptide from SAVINASE™(Novozymes) resulting in the coding sequence Sav-10RS which is shown inSEQ ID NO: 2. Several tail-variants of this construct were made.Compared to the Sav-10RS protease encoded by SEQ ID NO:2 the tailvariant construct Sav-10RS HV0 was constructed to have 8 amino acidsextra in the C-terminus: QSHVQSAP (SEQ ID NO: 3) which were encoded bythe following DNA sequence extension inserted in front of the TAAstopcodon of SEQ ID NO: 2:

(SEQ ID NO: 4): caatcgcatgttcaatccgctcca

Tail variant Sav-10RS HV1 was constructed to have 4 amino acids extra inthe C-terminus: QSAP (SEQ ID NO: 5), with the following DNA sequenceextension inserted in front of the TAA stopcodon:

(SEQ ID NO: 6): caatcggctcct

Tail variant Sav-10RS HV3 was constructed to have 2 amino acids extra inthe C-terminus: QP (SEQ ID NO: 7) with the following DNA sequenceextension inserted in front of the TAA stopcodon:

(SEQ ID NO: 8): caacca

Tail variant Sav-10RS HV2 was constructed to have one amino acid extrain the C-terminus: P (SEQ ID NO: 9) with the following DNA sequenceextension inserted in front of the TAA stopcodon:

(SEQ ID NO: 10): cca

The 10RS gene and the four tail-variant encoding genes were integratedby homologous recombination into the Bacillus subtilis MB1053 host cellgenome. Chloramphenicol resistant transformants were checked forprotease activity on 1% skim milk LB-PG agar plates (supplemented with 6μg/ml chloramphenicol). Some protease positive colonies were furtheranalyzed by DNA sequencing of the insert to ensure the correct gene DNAsequence, and five strains, each comprising one of the above constructs,were selected and denoted, respectively: B. subtilis Sav-10RS, B.subtilis Sav-10RS HV0, B. subtilis Sav-10RS HV1, B. subtilis Sav-10RSHV2 and B. subtilis Sav-10RS HV3.

Example 2 Fermentation Yields of 10R Tail-variants with Savinase Signal

Fermentations for the production of the tail-variant enzymes of theinvention were performed on a rotary shaking table in 500 ml baffledErlenmeyer flasks each containing 100 ml TY supplemented with 6 mg/lchloramphenicol.

Six Erlenmeyer flasks for each of the five B. subtilis strains fromexample 1 were fermented in parallel. Two of the six Erlenmeyer flaskswere incubated at 37° C. (250 rpm), two at 30° C. (250 rpm), and thelast two at 26° C. (250 rpm). A sample was taken from each shake flaskat day 1, 2 and 3 and analyzed for proteolytic activity. The results areshown in tables 1-3. As it can be seen from tables 1-3, the effect ofthe tails is a surprisingly high improvement on the expression level ofthe protease, as measured by activity in the culture broth. The effectis most pronounced at 26° C. and 30° C., but is also evident at 37° C.as an effect observed especially at the early stage of the fermentation.

TABLE 1 Relative proteolytic activities at 37° C. Day 1 Day 2 Day 3Sav-10RS 1.0 1.0 1.0 Sav-10RS HV0 3.3 0.7 0.8 Sav-10RS HV1 4.7 1.3 1.2Sav-10RS HV2 2.2 0.6 0.4 Sav-10RS HV3 5.3 1.4 1.7

TABLE 2 Relative proteolytic activities at 30° C. Day 1 Day 2 Day 3Sav-10RS 1.0 1.0 1.0 Sav-10RS HV0 1.7 2.2 2.9 Sav-10RS HV1 4.6 3.1 4.9Sav-10RS HV2 2.4 1.9 2.3 Sav-10RS HV3 4.8 3.0 4.4

TABLE 3 Relative proteolytic activities at 26° C. Day 1 Day 2 Day 3Sav-10RS 1.0 1.0 1.0 Sav-10RS HV0 1.8 2.5 3.1 Sav-10RS HV1 2.5 3.6 4.3Sav-10RS HV2 1.8 2.6 2.8 Sav-10RS HV3 2.6 3.5 4.6

Example 3 Chromosomal Integration of Tall-variant Genes

The following construct was used for the chromosomal integration of thetail-variant encoding genes. The coding sequence of the well-knownsubtilisin BPN′ protease was operationally linked to a triple promoter,a marker gene was fused to this (a spectinomycin resistance genesurrounded by resolvase res-sites), and pectate lyase encoding genesfrom Bacillus subtilis were fused to the construct as flanking segmentscomprising the 5′ polynucleotide region upstream[yfmD-ytmC-yfmB-yfmA-Pel-start], and the 3′ polynucleotide regiondownstream [Pel-end-yflS-citS(start)] of the tail-variant encodingpolynucleotide, respectively. The integrational cassette was made by thejoining of several different PCR fragments. After the final PCR reactionthe PCR product was used for transformation of naturally competent B.subtilis cells. One clone denoted PL3598-37 was selected and confirmedby sequencing to contain the correct construct.

The PL3598-37 Clone Thus Contains the Following:

1. The flanking regions 100% homologous to region of the B. subtilisgenome (appears as the upstream fragment yfmD-ytmC-yfmB-yfmA-Pelstartand the downstream fragment Pel-end-yflS-citS(start)).

2. The Spectinomycin resistance gene flanked by Resolvase sites (res).

3. The triple promoter region plus CryIIIA mRNA stabilising leadersequence.

4. The BPN′ Open Reading Frame.

Construction of Triple Promoter BPN′ Cassette

A PCR fragment comprising the integrational cassette for a BPN′ librarywas constructed, thus operably linking a triple promoter (as describedin WO 99/43835; Novozymes) to a BPN′ expression cassette from a Bacillusstrain. The triple promoter is a fusion of an optimized BacillusamyL-derived promoter (as shown in WO 93/10249; Novozymes) with twopromoters scBAN and cryIIIA, where the first is a consensus version ofthe Bacillus amyloliquefaciens amylase BAN promoter, and the latterincludes a mRNA-stabilising sequence (as described in WO 99/43835;Novozymes). Suitable primers can be derived from the publicly availablesequences (Vasantha, N. et al. Genes for alkaline protease and neutralprotease from Bacillus amyloliquefaciens contain a large open readingframe between the regions coding for signal sequence and mature protein.J. Bacteriol. 159:811 (1984) EMBL: accession No. K02496). A Kpnl and aSall restriction site was introduced to flank the PCR fragment at eachend, using the primers:

#252639 (SEQ ID NO: 11): catgtgcatgtgggtaccgcaacgttcgcagatgctgctgaagag#251992 (SEQ ID NO: 12): catgtgcatgtggtcgaccgattatggagcggattgaacatgcg

The Kpnl and Sall restriction sites in the PCR fragment weresubsequently used to clone the fragment into a Kpnl-Sall digestedPecl-Spec PCR fragment. The Pecl-Spec fragment comprises a Spectinomycinresistance gene inserted in the middle of the B. subtilis Pectate lyasegene plus approx. 2.3 kb of upstream genomic DNA and approx. 1.7 kbdownstream genomic DNA. The Pecl-Spec fragment was produced by PCRamplification of genomic DNA from the B. subtilis strain MB1053, usingthe primers:

#179541 (SEQ ID NO: 13): gcgttgagacgcgcggccgcgagcgccgtttggctgaatgatac#179542 (SEQ ID NO: 14): gcgttgagacagctcgagcagggaaaaatggaaccgctttttcConstruction of MB1053

The MB1053 B. subtilis strain was constructed by deletion of thepectatelyase (Pel) gene through integration of a PCR product into awild-type B. subtilis typestrain genome. This was achieved by a PCRamplification of genomic DNA directly downstream and upstream of thePectate lyase gene of the B. subtilis.

The ends of the genomic DNA directly preceding and proceeding the Pelgene were elongated through primer insertion of sequences being 100%homologous to DNA sequences defined by the ends of a third PCR fragmentencoding a marker gene surrounded by Resolvase (Res) sites. In thisparticular case the marker gene (Spec) conferred resistance tospectinomycin, and it was situated between two Res sites, altogetherpresent on the plasmid pSJ3358 (described In U.S. Pat. No. 5,882,888).Three different PCR fragments were initially produced.

-   Fragment 1: this fragment covers from the yfmD gene to the middle of    the Pel gene and introduces an overhang to the Res-Spec-Res cassette    at the Pel gene. The size of fragment 1 is 2.8 kb. The fragment was    produced by a PCR amplification chromosomal DNA from the B. subtilis    strain PL2306, using the primers:

#179541 (SEQ ID NO: 13), and #179539 with overlap to #179154 Spec primer(SEQ ID NO: 15): ccatttgatcagaattcactggccgtcgttttacaaccattgcggaaaatagtcataggcatcc

-   Fragment 2: this fragment covers from the middle of the Pel gene to    after the end of the CitS gene and introducing an overhang to the    Res-Spec-Res cassette at the middle of the Pel gene. The size of    fragment 2 is 2.3 kb. The fragment was produced by a PCR    amplification of chromosomal DNA from the B. subtilis strain PL2306,    using the primers:

#179542 (SEQ ID NO: 14), and #179540 with overlap to #179153 Spec primer(SEQ ID NO: 16): ggatccagatctggtacccgggtctagagtcgacgcggcggttcgcgtccggacagcaca

-   Fragment 3: this fragment contains the Spectinomycin gene surrounded    by Res sites and DNA sequences in the ends overlapping with PCR    fragment 1 and 2. The size of fragment 3 is 1.6 kb. Fragment 3 was    produced by PCR amplification of plasmid pSJ3358, using the primers:

#179154 (SEQ ID NO: 17): gttgtaaaacgacggccagtgaattctgatcaaatgg #179153(SEQ ID NO: 18): ccgcgtcgacactagacacgggtacctgatctagatcStandard Conditions for the PCR Reaction

For the PCR amplifications of fragment 1-3 the HiFi Expand™ PCR system(Roche) was used together with the following cycling scheme:

5 μl Buffer 2

14 μl dNTP's (1.25 mM each)

2.5 ud 20 μM primer 1

2.5 μl 2 μM primer 2

x μl water

To this mix 3 μl of DNA (apx. 100 ng) and 0.75 μl Enzyme mix (use hotstart) is added.

-   Total volume is 50 μl.-   The cycling profile is:    -   1 cycle of 120 sec at 94° C.        Break.

10 cycles of 15 sec at 94° C., 60 sec at 60° C., 240 sec at 72° C.

20 cycles of 15 sec at 94° C., 60 sec at 60° C., (180 sec at 72° C. add20 sec pr cycle)

1 cycle 600 sec at 68° C.

The three PCR fragments were made and joined in later JOINING-PCRreactions. The three PCR fragments were single sharp bands and no gelpurification was necessary. Only Qiagen™ PCR purification was performedprior to the following JOINING-PCR. JOINING of fragment 1+3 (sameprocedure for fragment 2+3):

5 μl Buffer 2

8 μl dNTP's (1.25 mM each)

5.0 μl Fragment 3

5.0 μl Fragment 1

9.25 μl water

1 cycle of 120 sec at 94° C.

Break. Add Enzyme

10 cycles of 15 sec at 94° C., 60 sec at 60° C., 240 sec at 72° C.

Break. Add Primers

15 cycles of 15 sec at 94° C., 60 sec at 60° C., (180 sec at 72° C. add20 sec pr cycle)

1 cycle 600 sec at 68° C.

After the first cycle at 94° C. for 120 sec there is a break, where 0.75μl Enzyme mix is added.

Total volume is now 45.0 μl.

After the initial 10 cycles, there is another break in the cycling andfor fragment 1+3: 2.5 μl (20 μM #179541) and 2.5 μl (20 μM #179153) areadded and for fragment 2+3: 2.5 μl (20 μM #179542) and 2.5 μl (20 μM#179154) are added and the cycling is continued for 15 cycles more.

The PCR products were then gel purified: The size of fragment 1+3 shouldbe 3.4 kb and the size of fragment 2+3 should be 3.4 kb. These twofragments were joined in a last PCR reaction (Expand™ long system,Roche):

5 μl Buffer 1

14 μl dNTP's (1.25 mM each)

5.0 μl Fragment 1+3

5.0 μl Fragment 2+3

17.75 μl water

After the first cycle at 94° C. for 120 sec there is a break, where 0.75μl Enzyme mix is added.

Total volume is now 45.0 μl.

After the initial 10 cycles, there is another break in the cycling and2.5 μl (20 μM #179541) and 2.5 μl (20 μM #179542) is added and thecycling is continued for 15 cycles more.

1 cycle of 120 sec at 94° C.

Break. Add Enzyme

10 cycles of 15 sec at 94° C., 60 sec at 60° C., 240 sec at 68° C.

Break. Add Primers

15 cycles of 15 sec at 94° C., 60 sec at 60° C., 180 sec at 68° C. add20 sec pr cycle

1 cycle 600 sec at 68° C.

The size of the joined PCR fragment is 6.8 kb. This PCR fragment waspurified using a Qiagen™ PCR purification kit, and 5 μl of the 50 μleluted DNA was used to transform a standard B. subtilis strain. Aftertransformation cells were spread onto LBPG-120 μg/ml of spectinomycin.Next day more than 1000 colonies were seen. 8 of these were checkedusing PCR primers from last JOINING PCR amplification yielding PCRfragment of 6.8 kb rather than the 5.2 kb expected if deletion had notoccurred. Furthermore, the pectatelyase activity of the clones waschecked with the Mancini Immunoassay, which showed no reactivity towardsthe pectatelyase activity. This taken together with the Spec resistancetells us that deletion had occurred. One such clone was selected anddenoted MB1053.

Insertion of BPN′ Expression Cassette Adjacent to the Res-spec-res inMB1053

The ligation mix of the digested PCR amplified triple promoter BPN′expression cassette and the Kpnl-Sal digested Pecl-Spec PCR fragment wasused as template in a PCR amplification using the PCR primers #179541and #179542. This resulted in a PCR fragment of approx. 9 kb, which wasused to transform B. subtilis PL1801 (Diderichsen, B et al. 1990.Cloning of aldB, which encodes alpha-acetolactate decarboxylase, anexoenzyme from Bacillus brevis. J. Bacteriol., 172, 4315-4321) competentcells. The transformed cells were plated on LB-120 μg/ml Spectinomycinagar plates with skim milk. Spectinomycin resistant colonies with largeskim milk clearing zones were restreaked on Spectinomycin agar platesand analysed for the integration of the PCR fragment with PCR using theprimers #179541 (SEQ ID NO: 13) and #179542 (SEQ ID NO: 14).

Appearance of a 9 kb fragment indicates that the PCR fragment has beenintegrated into the host cell genome. Several of these clones weresequenced to confirm integration of the expression cassette, one suchclone was selected and denoted PL3598-37.

Example 4 Construction of Plasmid-Borne Chromosomal IntegrationalCassette

An E. coli plasmid-borne integrational cassette for a library may beconstructed In vivo. An integration cassette to be used according to themethod of the invention may be present on a E. coli plasmid (which iscapable only of replication in E. coli, not in B. subtilis), the plasmidcomprising:

i) The DNA sequence encoding the Pre-Pro-domains of the subtilisinprotease commonly known as Savinase, preceded by and operably linked to

ii) a DNA sequence comprising a mRNA stabilising segment derived in thisparticular case from the CryIIIa gene;

iii) a marker gene (a chloramphenicol resistance gene), and

iv) genomic DNA from Bacillus subtilis as 5′ and 3′ flanking segments:The homologous 5′ polynucleotide region upstream of the polynucleotide[yfmD-ytmC-yfmB-yfmA-Pel-start], and the 3′ polynucleotide regiondownstream of the polynucleotide [Pel-end-yflS-citS(start)],respectively.

The cassette was made by several cloning steps involving digestion ofpUC19 plasmid and PCR fragments with appropriate restrictionendonuclease sites of several different PCR fragments in the generallyused plasmid pUC19. After each ligation of a PCR fragment into aplasmid, the ligation mixture was transformed into electrocompetentDH5alpha E. coli cells that were prepared for and transformed byelectroporation using a Gene Pulser™ electroporator from BIO-RAD asdescribed by the supplier. One final plasmid construct was confirmed bysequencing to contain the correct construct as outlined above, and itwas denoted pMB1508.

The pMB1508 Plasmid Thus Contains the Following:

i) The CryIIIA mRNA stabilising leader sequence including a ribosomebinding sequence (RBS), operationally linked to

ii) DNA encoding the Pre-Pro-domains of the subtilisin commonly known asSavinase, including Kpnl and Notl sites for cloning;

iii) The chloramphenicol resistance operon;

iv) The 3′ downstream flanking region [Pel-end-yflS-citS(start)] whichis 99-100% homologous to the region of the B. subtilis.

The four elements listed were cloned in the pUC19 vector (Isolated fromE. coli ATCC 37254; Vieira J, Messing J. The pUC plasmids, anM13mp7-derived system for insertion mutagenesis and sequencing withsynthetic universal primers. Gene 19: 259-268, 1982.) in the EcoRI andSall sites to give pMB1 508. In order for the resulting plasmid tointegrate effeciently to a specified site of th B. subtilis genome, anew strain was established. The new strain is a derivative of Bacillussubtilis 168 BGSC accession number: 1A1 168 trpC2. The strain was madecompetent and transformed as described above. Using elements from thePL3598-37 clone described above, the new integration strain denotedMB1510 was established and characterised to contain the followingelements from PL3598-37:

i) The triple promoter and the mRNA stabilising element.

ii) Flanking segments comprising the following homologous polynucleotideregion [yfmD-ytmC-yfmB-yfmA-Pel-start] upstream of the triple-promoter,and the polynucleotide region [Pel-end-yflS-citS(start)] downstream ofthe mRNA stabilizing element.

Thus, when using MB1510 competent cells, it is possible for the pMB1508(or derivatives thereof) to directly integrate into the genome of MB1510where the two flanking regions in fusion with the triple-promoter andmRNA stabilising element is located, resulting in a construction wherethe incoming PrePro encoding DNA of pMB1508DNA has been integrated inthe correct reading frame with the tripel-promoter, the mRNA stabilisingelement and the RBS. Thus resulting in high expression of the integratedgene from the promoter elements already present on the genome of MB1510.

Transformation effeciency was established for the B. subtilis strainMB1510 transformed with E. coli prepared plasmid pMB1508. For furthertesting of the potential of using this approach, the Savinase encodinggene of Bacillus clausii was PCR amplified using the two PCR primers:

Primer #317 (SEQ ID NO: 19) tggcgcaatcggtaccatgggg Primer #139 NotI (SEQID NO: 20) catgtgcatgcggccgcattaacgcgttgccgcttctgcg

The resulting ˜0.8 kb of the Savinase fragment and the pMB1508 plasmidare digested with Kpnl and Notl, and the resulting fragments are thenpurifiied by agarose gel electrophoresis. The two fragments are ligated,and the ligation mixture is used to transform competent E. coli cellswhich are then plated on LB-agar plates or placed in liquid media forgrowth overnight at 37° C.; both types of media containing 50-100 μg/mlof Ampicillin. After incubation, a plasmid prep is made of the liquidculture. The purified plasmid is used for transformation of competentcells of MB1510 (using 100-10.000 ng of plasmid per transformation. Thetransformed cells are plated onto TY medium with 2% skimmilk and 6 μg/mlof chloramphenicol for selection. After overnight incubation at 37° C.clearing zones appear around those colonies wherein the integrationcassette is integrated properly into the cells, indicating high Savinaseexpression.

This approach can also be used to make highly diverse libraries of anygene of interest expressable in B. subtilis, where rather than a geneencoding one enzyme, any expressable polynucleotide is inserted into theplasmid pMB1508 and integrated into the MB1510 strain for subsequentscreening.

Sequence of Plasmid pMB1508 (SEQ ID NO: 21)

The Plasmid pMB1508 has the Following Components, Indicated by BasepairPositions:

BP 5186-395: pUC19 sequence from E. coli clone ATCC 37254, Vieira J,Messing J. The pUC plasmids, an M13mp7-derived system for insertionmutagenesis and sequencing with synthetic universal primers. Gene 19:259-268, 1982.

BP 396-1021: EcoR I cloning site (BP396-401) and the CryIIIA mRNAstabilising element. (Described in WO 9634963-A1)

BP 1022-1412: Encodes the Pre-Pro sequence of Savinase and the NotIcloning site. (Pre-Pro part described in eg. WO 9623073-A1, the NotIsite and the spacing between the Pre-Pro and NotI was introduced by thePCR primer.

BP 1413-2512: The BgI II cloning site (BP1413-1418) and theChloramphenicol acetyl-transferase operon of pDN1050 (Described in eg.Diderichsen, B.; Poulsen, G. B.; Joergensen, S. T.; A useful cloningvector for Bacillus subtilis . Plasmid 30:312 (1993)).

BP 2513-5185: The polynucleotide region [Pel-end-yflS-citS(start)]downstream of the pelB locus of the B. subtilis genome. (as it appeaarsfrom the publication and corresponding database of: F. Kunst, N.Ogasawara, I. Moszer, <146 other authors>, H. Yoshikawa, A. Danchin.“The complete genome sequence of the Gram-positive bacterium Bacillussubtilis ” Nature (1997) 390:249-256).

The Bacillus subtilis Strain MB1510

MB1510 has the Following Specific Features in and Around the pelB Locus:

-   i) The triple promoter and the mRNA stabilising element including a    RBS (Ribosome binding sequence).-   ii) Flanking segments comprising the following homologous    polynucleotide region [yfmD-ytmC-yfmB-yfmA-Pel-start] upstream of    the triple-promoter, and the polynucleotide region    [Pel-end-yflS-citS(start)] downstream of the mRNA stabilizing    sequence.    Sequence of MB1551 Genomic Integration Region (SEQ ID NO: 22)

BP 1-2873: corresponds to sequence of Bacillus subtilis genomeyfmD-ytmC-yfmB-yfmA-Pel-start (as it appeaars from the publication andcorresponding database of: F. Kunst et al. “The complete genome sequenceof the Gram-positive bacterium Bacillus subtilis” Nature (1997)390:249-256).

BP 3102-4082: The triple promoter and CryIIIA mRNA stabilising elementplus RBS. (Described above in PL3598-37 construct).

BP 4083-5718: The polynucleotide region [Pel-end-yflS-citS(start)] endof and downstream of the pelB locus of the B. subtilis genome (as itappeaars from the publication and corresponding database of. F. Kunst,N. Ogasawara, I. Moszer, <146 other authors>, H. Yoshikawa, A. Danchin.“The complete genome sequence of the Gram-positive bacterium Bacillussubtilis” Nature (1997) 390:249-256).

Example 5 Construction of a 2 Amino-acid Tail-variant Library

This example shows the construction of a tail-variant library. In thislibrary two amino acids were introduced at the C-terminal of the 10Rprotein. Such a Tail-library may be made with the method described aboveusing the following PCR primers in a PCR reaction using genomic DNA fromB. subtilis 10RS as template:

1605 (SEQ ID NO: 23): gacggccagtgaattcgataaaagtgc 1606 (SEQ ID NO: 24):ccagatctctatnktnktgtacggagtctaactccccaagagwherein N=A, C, G or T; and K=T or G.

The resulting PCR product was digested with EcoR I and BgI II andligated into EcoR I and BgI II digested pMB1508. Hereafter following theprinciple described above.

Chloramphenicol resistant Bacillus subtilis transformants were picked bya robotic colony picker from a bioassay plate and transferred into a 384well microtiter plate (MTP) containing 0.05×TY supplemented with 6 mg/lchloramphenicol (60 μl/well). The MTPs were incubated at 26° C. for 72h. After incubation each well was analyzed for proteolytic activity.

The thirty Bacillus subtilis transformants with highest proteolyticactivity were selected for determination of the two tail amino acids ineach transformant by DNA sequencing, the sequencing results aresummaries in table 4 and table 5.

TABLE 4 column one shows the amino acid sequence of the tail, and columntwo shows the number of Bacillus subtilis transformants sequenced withthat particular AA tail sequence. AA Tail No. of transformants TL 4 TT 4QL 3 TP 3 LP 3 TI 2 IQ 2 QP 2 PI 2 LT 1 TQ 1 IT 1 QQ 1 PQ 1 Total 30

TABLE 5 The table shows the amino acid which could be introduced by theprimer used for the library construct and the actual findings by DNAsequencing of the thirty colonies isolated from screening. Possibilitiesposition 1 Result Possibilities position 2 Result K 0 K 0 R 0 R 0 T 14 T6 I 3 I 4 Q 6 Q 5 P 3 P 8 L 4 L 7 Total 30 Total 30

Example 6 Construction of Bacillus subtilis Strains L2, L2 HV0, and L2HV1

A Bacillus subtilis strain was made analogously with the construction ofthe Bacillus subtilis strain 10RS, with the DNA coding for the pro-formof the S2A protease from Nocardiopsis dassonvillei subsp. DassonvilleiDSM 43235, denoted L2, fused by PCR in frame to the DNA coding for thesignal peptide from SAVINASE™ (a well-known commercial protease derivedfrom Bacillus clausii, available from Novozymes, Denmark), the resultingstrain was denoted Bacillus subtilis Sav-L2.

The DNA sequence including the coding region for the pro-mature S2Aprotease from Nocardiopsis dassonvillei subsp. Dassonvillei DSM 43235,as amplified with primers 1423 and 1475, is shown in SEQ ID NO: 25. Thecorresponding encoded pro-form amino acid sequence for the L2 proteaseis shown in SEQ ID NO: 28.

1423 (SEQ ID NO: 26): gcttttagttcatcgatcgcatcggctgctccggcccccgtcccccag1475 (SEQ ID NO: 27): ggagcggattgaacatgcgattaggtccggatcctgacaccccag

Two tail-variants of this construct were also made. Tail variant Sav-L2HV0 was constructed to have 8 amino acids extra in the C-terminus:QSHVQSAP (SEQ ID NO: 3), by using the DNA sequence extension inserted infront of the TAA stopcodon which is shown in SEQ ID NO: 4. Tail variantSav-L2 HV1 was constructed to have 4 amino acids extra in theC-terminus: QSAP (SEQ ID NO: 5), by using the DNA sequence extensioninserted in front of the TAA stopcodon which is shown in SEQ ID NO: 6.Both tail variants had the SAVINASE™ signal-peptide encoding sequencefused in frame with the pro-mature encoding sequence, just like inSav-L2.

The Sav-L2 gene and the two tail-variants Sav-L2 HV0 and Sav-L2 HV1 wereintegrated by homologous recombination on the Bacillus subtilis MB1053host cell genome as outlined above. Chloramphenicol resistanttransformants were checked for protease activity on 1% skim milk LB-PGagar plates (supplemented with 6 μg/ml chloramphenicol). Some proteasepositive colonies were further analyzed by DNA sequencing of the insertto confirm the correct DNA sequence, and one strain for each constructwas selected and denoted B. subtilis Sav-L2, B. subtilis Sav-L2 HV0, andB. subtilis Sav-L2 HV1, respectively.

Example 7 Fermentation Yields of the Bacillus Strains of Example 6

The three B. subtilis strains of example 6, were fermented on a rotaryshaking table in 500 ml baffled Erlenmeyer flasks containing 100 ml TYsupplemented with 6 mg/l chloramphenicol. Six Erlenmeyer flasks for eachof the three B. subtilis strains were fermented in parallel. Two of thesix Erlenmeyer flasks were incubated at 37° C. (250 rpm), two at 30° C.(250 rpm), and the last two at 26° C. (250 rpm). A sample was taken fromeach shake flask at day 1, 2 and 3 and analyzed for proteolyticactivity. The results are shown in tables 6-8. As it can be seen fromtables 6-8, the effect of the tails also increases the expression levelfor the Sav-L2 protease from Nocardiopsis dassonvillei subsp.Dassonvillei DSM 43235 when expressed in B. subtilis. An increase of upto 40% is observed in this experiment, but overall improvement isobserved for both tail-variants at all three temperatures tested.

TABLE 6 Relative proteolytic activities at 37° C. Day 1 Day 2 Day 3Sav-L2 1.0 1.0 1.0 Sav-L2 HV1 1.4 1.3 1.2 Sav-L2 HV0 1.3 1.1 1.4

TABLE 7 Relative proteolytic activities at 30° C. Day 1 Day 2 Day 3Sav-L2 1.0 1.0 1.0 Sav-L2 HV1 1.0 1.2 1.4 Sav-L2 HV0 1.1 1.3 1.3

TABLE 8 Relative proteolytic activities at 26° C. Day 1 Day 2 Day 3Sav-L2 1.0 1.0 1.0 Sav-L2 HV1 1.3 1.1 1.1 Sav-L2 HV0 0.2 1.1 1.1

Example 8 10R Tail-variants with Heterologous Pro-regions in Bacillus

The DNA sequence coding for the pro-region from the L2 protease fromNocardiopsis dassonvillei subsp. Dassonvillei, DSM 43235 is shown in SEQID NO: 29, and the corresponding amino acid sequence is shown in SEQ IDNO: 30. A Bacillus subtilis strain denoted L210R, similar to theBacillus subtilis strain 10RS, but with the DNA coding for thepro-region of the L2 replacing the pro-region of 10RS, was made. Theentire L210R protease encoding sequence incl. the pro-region of L2, isshown in SEQ ID NO: 31.

Two tail variants of the above construct were also made. Tail variantHV0 was constructed to have 8 amino acids extra in the C-terminus:QSHVQSAP (SEQ ID NO: 3) with the DNA shown in SEQ ID NO: 4 inserted infront of the TAA stopcodon of the encoding sequence. Tail variant HV1was constructed to have 4 amino acids extra in the C-terminus: QSAP (SEQID NO: 5) with the DNA sequence shown in SEQ ID NO: 6 inserted in frontof the TAA stopcodon of the encoding sequence.

The 10RL2) construct and the two tail variants were integrated byhomologous recombination on the Bacillus subtilis MB1053 host cellgenome. Chloramphenicol resistant transformants were checked forprotease activity on 1% skim milk LB-PG agar plates (supplemented with 6μg/ml chloramphenicol). Some protease positive colonies were furtheranalyzed by DNA sequencing of the insert to confirm the correct DNAsequence, and a strain for each construct was selected, and denoted B.subtilis L210R, B. subtilis L210R HV0, and B. subtilis L210R HV1,respectively.

Example 9 Fermentation Yields of 10R Tail-variants with HeterologousPro-region

The six B. subtilis strains 10RS, 10RS HV0, 10RS HV1, L210R, L210R HV0,and L210R HV1 ₁, were fermented on a rotary shaking table in 500 mlbaffled Erlenmeyer flasks containing 100 ml TY supplemented with 6 mg/lchloramphenicol. Six Erlenmeyer flasks for each of the B. subtilisstrains were fermented in parallel. Two of the six Erlenmeyer flaskswere incubated at 37° C. (250 rpm), two at 30° C. (250 rpm), and thelast two at 26° C. (250 rpm). A sample was taken from each shake flaskat day 1, 2 and 3 and analyzed for proteolytic activity. The results areshown in FIG. 1, and in tables 9-11. As it can be seen from the results,the effect of the exchange of the proregion from 10R with the proregionfrom the L2 protease resulted in a surprisingly high improvement on theexpression level of the 10R protease as measured by proteolytic activityin the culture broth at 37° C. The effect is most pronounced in the twotail variants.

TABLE 9 Relative proteolytic activities at 37° C. Day 1 Day 2 Day 3 10RS1.0 1.0 1.0 10RS HV0 3.7 8.9 3.5 10RS HV1 3.9 8.5 4.3 L210R 1.9 2.3 1.6L210R HV0 5.3 14.4 7.3 L210R HV1 9.1 20.9 7.6

TABLE 10 Relative proteolytic activities at 30° C. Day 1 Day 2 Day 310RS 1.0 1.0 1.0 10RS HV0 2.8 3.1 4.3 10RS HV1 3.6 3.6 4.9 L210R 0.6 0.40.9 L210R HV0 3.5 3.2 4.5 L210R HV1 3.7 3.2 4.5

TABLE 11 Relative proteolytic activities at 26° C. Day 1 Day 2 Day 310RS 1.0 1.0 1.0 10RS HV0 2.6 3.0 2.8 10RS HV1 3.7 3.3 3.1 L210R 0.4 0.70.4 L210R HV0 2.3 2.1 1.9 L210R HV1 2.2 1.7 1.7

Example 10 Repeat of Examples 1-9 with other 10R-like Proteases

Completely analogously with the above examples 1 through 9, similarexperiments are carried out with the proteases of the followingNocardiopsis strains:

-   (a) Nocardiopsis dassonvillei NRRL 18133 as described in WO    88/03947;-   (b) Nocardiopsis sp. NRRL 18262 as described in WO 88/03947, the DNA    and amino acid sequences of the protease derived from Nocardiopsis    sp. NRRL 18262 are shown in DK patent application no. 1996 00013,    and WO 01/58276 describes the use in animal feed of acid-stable    proteases related to the protease derived from Nocardiopsis sp. NRRL    no. 18262;-   c) Nocardiopsis Alba DSM 15647; the amino acid sequence of the    protease is SEQ ID NO: 33, the encoding nucleotide sequence is SEQ    ID NO: 32; the gene is isolated from the genomic DNA of this strain    by PCR-amplification using the two primers:

1421 (SEQ ID NO: 34): gttcatcgatcgcatcggctgcgaccggccccctcccccagtc 1604(SEQ ID NO: 35): gcggatcctatcaggtgcgcagggtcagacc.

-   (d) Nocardiopsis prasina DSM 15648; the amino acid sequence of the    protease is SEQ ID NO: 37, the encoding nucleotide sequence is SEQ    ID NO: 36; the gene is isolated from the genomic DNA of this strain    by PCR-amplification using the two primers:

1346 (SEQ ID NO: 38): gttcatcgatcgcatcggctgccaccggaccgctcccccagtc 1602(SEQ ID NO: 39): gcggatcctattaggtccggagacggacgccccaggag.

-   (e) Nocardiopsis prasina DSM 15649; the amino acid sequence of the    protease is SEQ ID NO: 41, the encoding nucleotide sequence is SEQ    ID NO: 40; the gene is isolated from the genomic DNA of this strain    by PCR-amplification using the two primers:

1603 (SEQ ID NO: 42): gttcatcgatcgcatcggctgccaccggaccactcccccagtc, and1602 (SEQ ID NO: 39).

Example 11 In vivo Monogastric Performance of a 10R-like Protease fromDSM 43235

The performance of the Nocardiopsis dassonvillei subspecies dassonvilleiDSM 43235 protease assayed in a monogastric in vitro digestion model.The performance of a purified preparation of the mature part of theprotease having SEQ ID NO: 28 (prepared as described above) was testedin an in vitro model simulating the digestion in monogastric animals. Inparticular, the protease was tested for its ability to improvesolubilisation and digestion of maize/-SBM (maize/-soybean meal)proteins. In the tables below, this protease is designated “protease ofthe invention.”

The in vitro system consisted of 15 flasks in which maize/-SBM substratewas initially incubated with HCl/pepsin—simulating gastric digestion—andsubsequently with pancreatin—simulating intestinal digestion. 10 of theflasks were dosed with the protease at the start of the gastric phasewhereas the remaining flasks served as blanks. At the end of theintestinal incubation phase samples of in vitro digesta were removed andanalysed for solubilised and digested protein.

TABLE 12 Outline of in vitro digestion procedure Simulated Timedigestion Components added pH Temperature course phase 10 g maize/-SBMsubstrate 3.0 40° C. t = 0 min Mixing (6:4), 41 ml HCl (0.105M) 5 ml HCl(0.105M)/pepsin 3.0 40° C. t = 30 min Gastric (3000 U/g substrate), 1 mLdigestion protease of the invention 16 ml H₂O 3.0 40° C. t = 1.0 hourGastric digestion 7 ml NaOH (0.39M) 6.8 40° C. t = 1.5 hours Intestinaldigestion 5 ml NaHCO₃ (1M)/ 6.8 40° C. t = 2.0 hours Intestinalpancreatin (8 mg/g diet) digestion Terminate incubation 7.0 40° C. t =6.0 hoursConditions

-   Substrate: 4 g SBM, 6 g maize (premixed)-   pH: 3.0 stomach step/6.8-7.0 intestinal step-   HCl: 0.105 M for 1.5 hours (i.e. 30 min HCl-substrate premixing)-   pepsin: 3000 U/g diet for 1 hour-   pancreatin: 8 mg/g diet for 4 hours-   temperature: 40° C.-   Replicates: 5    Solutions-   0.39 M NaOH-   0.105 M HCl-   0.105 M HCl containing 6000 U pepsin per 5 ml-   1 M NaHCO₃ containing 16 mg pancreatin per ml-   125 mM NaAc-buffer, pH 6.0    Enzyme Protein Determinations

The amount of protease enzyme protein (in what follows, Enzyme Proteinis abbreviated EP) is calculated on the basis of the A₂₈₀ values and theamino acid sequences (amino acid compositions) using the principlesoutlined in S. C. Gill & P. H. von Hippel, Analytical Biochemistry 182,319-326, (1989).

Experimental Procedure for in vitro Model

The experimental procedure was according to the above outline. pH wasmeasured at time 1, 2.5, and .5 hours. Incubations were terminated after6 hours and samples of 30 ml were removed and placed on ice beforecentrifugation (10000×g, 10 min, 4° C.). Supernatants were removed andstored at −20° C.

Analysis

All samples were analysed for % degree of protein with the OPA method aswell as content of solubilised and digested protein using gelfiltration.

DH Determination by the OPA-method

The Degree of Hydrolysis (DH) of protein in different samples wasdetermined using an semi-automated microtiter plate based colorimetricmethod (Nielsen, P. M.; Petersen, D.; Dambmann, C. Improved method fordetermining food protein degree of hydrolysis. J. Food Sci. 2001, 66,642-646). The OPA reagent was prepared as follows: 7.620 g di-Natetraborate decahydrate and 200 mg sodiumdodecyl sulphate (SDS) weredissolved in 150 ml deionized water. The reagents were completelydissolved before continuing. 160 mg o-phthal-dialdehyde 97% (OPA) wasdissolved in 4 ml ethanol. The OPA solution was transferredquantitatively to the above-mentioned solution by rinsing with deionizedwater. 176 mg dithiothreitol 99% (DTT) was added to the solution thatwas made up to 200 ml with deionized water. A serine standard (0.9516meqv/l) was prepared by solubilising 50 mg serine (Merck, Germany) in500 ml deionized water.

The sample solution was prepared by diluting each sample to anabsorbance (280 nm) of about 0.5. Generally, supernatants were diluted(100×) using an automated Tecan dilution station (Männedorf,Switzerland). All other spectrophotometer readings were performed at 340nm using deionized water as the control. 25 μl of sample, standard andblind was dispensed into a microtiter plate. The micro-titer plate wasinserted into an iEMS MF reader (Labsystems, Finland) and 200 μl of OPAreagent was automatically dispensed. Plates were shaken (2 min; 700 rpm)before measuring absorbance. Finally, the DH was calculated. Eightfolddetermination of all samples was carried out.

Estimation of Solubilised and Digested Protein

The content of solubilised protein in supernatants from in vitrodigested samples was estimated by quantifying crude protein (CP) usinggel filtration HPLC. Supernatants were thawed, filtered through 0.45 μmpolycarbonate filters and diluted (1:50, v/v) with H₂O. Diluted sampleswere chromatographed by HPLC using a Superdex Peptide PE (7.5×300 mm)gel filtration column (Global). The eluent used for isocratic elutionwas 50 mM sodium phosphate buffer (pH 7.0) containing 150 mM NaCl. Thetotal volume of eluent per run was 26 ml and the flow rate was 0.4ml/min. Elution profiles were recorded at 214 nm and the total areaunder the profiles was determined by integration. To estimate proteincontent from integrated areas, a calibration curve (R²=0.9993) was madefrom a dilution series of an in vitro digested reference maize/-SBMsample with known total protein content. The protein determination inthis reference sample was carried out using the Kjeldahl method(determination of % nitrogen; A.O.A.C. (1984) Official Methods ofAnalysis 14th ed., Washington D.C.).

The content of digested protein was estimated by integrating thechromatogram area corresponding to peptides and amino acids having amolecular mass of 1500 Dalton or below (Savoie, L.; Gauthier, S. F.Dialysis Cell For The In-vitro Measurement Of Protein Digestibility. J.Food Sci. 1986, 51, 494-498; Babinszky, L.; Van, D. M. J. M.; Boer, H.;Den, H. L. A. An In-vitro Method for Prediction of The Digestible CrudeProtein Content in Pig Feeds. J. Sci. Food Agr. 1990, 50, 173-178;Boisen, S.; Eggum, B. O. Critical Evaluation of In-vitro Methods forEstimating Digestibility in Simple-Stomach Animals. Nutrition ResearchReviews 1991, 4, 141-162). To determine the 1500 Dalton dividing line,the gel filtration column was calibrated using cytochrome C (Boehringer,Germany), aprotinin, gastrin I, and substance P (Sigma Aldrich, USA), asmolecular mass standards.

Results

The results shown in Tables 13 and 14 below indicate that the proteaseincreased the Degree of Hydrolysis (DH), as well as soluble anddigestible protein significantly.

TABLE 13 Degree of Hydrolysis (DH), absolute and relative values EnzymeOf total (dosage in mg protein Relative to blank EP/kg feed) n % DH SD %DH % CV Blank 5 26.84 ^(a) 0.69 100.0 ^(a) 2.57 Protease of theinvention 5 28.21 ^(b) 0.35 105.1 ^(b) 1.25 (100) Different letterswithin the same column indicate significant differences (1-way ANOVA,Tukey-Kramer test, P < 0.05). SD = Standard Deviation. % CV =Coefficient of Variance = (SD/mean value) × 100%

TABLE 14 Solubilised and digested crude protein measured by ÄKTA HPLC.Enzyme Of total protein Relative to blank (dosage in % % % % mg EP/kgdig. sol. dig. sol. feed) n CP SD CP SD CP CV % CP CV % Blank 5 54.1^(a) 1.1 90.1 ^(a) 1.1 100.0 ^(a) 2.0 100.0 ^(a) 1.2 Protease of theinvention  (50) 5 57.7 ^(b) 1.1 93.2 ^(b) 1.4 106.7 ^(b) 1.9 103.4 ^(b)1.5 (100) 5 58.9 ^(b) 0.8 94.8 ^(b) 0.9 108.9 ^(b) 1.3 105.2 ^(b) 0.9Different letters within the same column indicate significantdifferences (1-way ANOVA, Tukey-Kramer test, P < 0.05). SD = StandardDeviation. % CV = Coefficient of Variance = (SD/mean value) × 100%

Example 12 In vitro Aquaculture Performance of 10R-like Protease fromDSM 43235

Performance of the protease from Nocardiopsis dassonvillei subsp.dassonvillei DSM 43235 in an aquaculture in vitro model. The proteasepreparation as described in Example 3 was tested in an aquaculture invitro model simulating the digestion in coldwater fish. The in vitrosystem consisted of 15 flasks in which SBM substrate was initiallyincubated with HCl/pepsin—simulating gastric digestion—and subsequentlywith pancreatin—simulating intestinal digestion. 10 of the flasks weredosed with the protease at the start of the gastric phase whereas theremaining 5 flasks served as blanks. At the end of the intestinalincubation phase samples of in vitro digesta were removed and analysedfor solubilised and digested protein.

TABLE 15 Outline of aqua in vitro digestion procedure Tem- Simulatedpera- Time digestion Components added pH ture course phase 10 g extrudedSBM substrate, 3.0 15° C. t = 0 min Gastric 62 mL HCl (0.155M)/pepsindigestion (4000 U/g substrate), 1 mL of the protease of the invention 7mL NaOH (1.1M) 6.8 15° C. t = 6 hours Intestinal digestion 5 mL NaHCO₃(1M)/pancreatin 6.8 15° C. t = 7 hours Intestinal (8 mg/g diet)digestion Terminate incubation 7.0 15° C. t = 24 hoursConditions

-   Substrate: 10 g extruded SBM-   pH: 3.0 stomach step/6.8-7.0 intestinal step-   HCl: 0.155 M for 6 hours-   Pepsin: 4000 U/g diet for 6 hours-   Pancreatin: 8 mg/g diet for 17 hours-   Temperature: 15° C.-   Replicates: 5    Solutions-   1.1 M NaOH-   0.155 M HCl/pepsin (4000 U/g diet)-   1 M NaHCO₃ containing 16 mg pancreatin/mL-   125 mM NaAc-buffer, pH 6.0    Experimental Procedure for Aqua in vitro Model

The experimental produce was according to the above outline. pH wasmeasured at time 1, 5, 8 and 23 hours. Incubations were terminated after24 hours and samples of 30 mL were removed and placed on ice beforecentrifugation (13000×g, 10 min, 0° C.). Supernatants were removed andstored at −20° C.

Analysis

-   All supernatants were analysed using the OPA method (% degree of    hydrolysis) and by ÄKTA HPLC to determine solubilised and digested    protein (see monogastric example).    Pre-treatment of in vitro Supernatants with EASY SPE Columns

Before analysis on ÄKTA HPLC supernatants from the in vitro system werepretreated using solid-phase sample purification. This was done toimprove the chromatography and thereby prevent unstable elution profilesand baselines. The columns used for extraction were solid phaseextraction columns (Chromabond EASY SPE Columns from Macherey-Nagel). 2mL milliQ water was eluted through the columns by use of a vacuumchamber (vacuum 0.15×100 kPa). Subsequently 3 mL in vitro sample wasdispensed onto the column and eluted (vacuum 0.1×100 kPa), the first ½mL of eluted sample was thrown away and a clean tube was placed beneaththe column, then the rest of the sample was eluted and saved for furtherdilution.

Results

The results shown in Tables 16 and 17 below indicate that the proteasesignificantly increased Degree of hydrolysis and protein digestibility.

TABLE 16 Degree of Hydrolysis (DH) measured by the OPA method, absoluteand relative values Of total Enzyme protein Relative to blank (mg EP/kgdiet) n % DH SD % DH % CV Blank 5 21.30 ^(a) 0.52 100.0 ^(a) 2.42Protease of the invention 5 21.98 ^(b) 0.22 103.2 ^(b) 1.00 (50)Different letters within the same column indicate significantdifferences (1-way ANOVA, Tukey-Kramer test, P < 0.05). SD = StandardDeviation. % CV = Coefficient of Variance = (SD/mean value) × 100%

TABLE 17 Solubilised and digested crude protein measured by ÄKTA HPLC,absolute and relative values Enzyme Of total protein Relative to blank(mg EP/kg % CP % CP % CP % CP diet) N dig SD sol SD dig % CV sol % CVBlank 5 50.0 ^(a) 2.2 89.9 ^(a) 3.2 100.0 ^(a) 4.5 100.0 ^(a) 3.5Protease of the invention  (50) 5 52.3 ^(b) 1.1 91.4 ^(a) 1.5 104.8 ^(b)2.1 101.7 ^(a) 1.6 (100) 5 53.4 ^(b) 0.4 91.6 ^(a) 1.0 107.0 ^(b) 0.7101.9 ^(a) 1.1 Different letters within the same column indicatesignificant differences (1-way ANOVA, Tukey-Kramer test, P < 0.05). SD =Standard Deviation. % CV = Coefficient of Variance = (SD/mean value) ×100%.

Example 13 Fermentation and Activity of 10R Tall-variants TQ and TP withSavinase Signal

Two of the B. subtilis strains of Example 5, strain 209 with the aminoacid tail-variant TQ, and strain 211 with the tail-variant TP, togetherwith B. subtilis Sav-10RS, were fermented on a rotary shaking table in500 ml baffled Erlenmeyer flasks containing 100 ml TY supplemented with6 mg/l chloramphenicol. Twelve Erlenmeyer flasks for each of the threeB. subtilis strains were fermented in parallel. Four of the twelveErlenmeyer flasks were incubated at 37° C. (250 rpm), four at 30° C.(250 rpm), and the last four at 26° C. (250 rpm). A sample was takenfrom each shake flask at day 1, 2 and 3 and analyzed for proteolyticactivity. The results are shown in tables 18 to 20 below.

As it can be seen from tables below, the effect of the 2 amino acidtails is a surprisingly high improvement on the yield of the protease,as measured by activity in the culture broth. The effect of the 2 aminoacid tails is comparable to the effect observed for Sav-10RS HV1 andSav-10RS HV3 in Example 1.

TABLE 18 Relative proteolytic activities at 37° C. 1 2 3 10R synt-15 1.01.0 1.0 209 7.0 7.0 6.0 211 7.2 7.7 4.9

TABLE 19 Relative proteolytic activities at 30° C. 1 2 3 10R synt-15 1.01.0 1.0 209 4.5 3.6 4.9 211 4.0 4.1 5.0

TABLE 20 Relative proteolytic activities at 26° C. 1 2 3 10R synt-15 1.01.0 1.0 209 6.4 4.3 4.0 211 3.7 4.1 4.2

Example 14 Synthetic Shuffled 10R-like Protease Tail-variants withSignal

A synthetic tail variant 10R protease encoding gene, denoted G-MAT-22,was constructed with a signal peptide, and the 8 amino acid C-terminaltail of SEQ ID NO: 3, and introduced into a Bacillus host forexpression. A surprisingly high yield of protease was achieved (data notshown). The full coding DNA sequence of G-MAT-22 is shown in SEQ ID NO:44, and the encoded pre-pro-protease is shown in SEQ ID NO: 45. TheG-mat-22 protease is an alpha-lytic protease-like enzyme (peptidasefamily S1E—old notation: S2A). This protease has a higher temperatureoptimum (at pH 9) than the 10R protease, as shown in FIG. 1.

Example 15 Shuffled Pro-sequences of 10R-like Proteases

Recombination of protease genes can be made independently of thespecific sequence of the parents by synthetic shuffling as described inNess, J. E. et al 2002 [Nature Biotechnology, Vol. 20 (12), pp.1251-1255, 2002]. Synthetic oligonucleotides degenerated in their DNAsequence to provide the possibility of all amino acids found in the setof parent proteases are designed and the genes assembled according tothe reference. The shuffling can be carried out for the full lengthsequence or for only part of the sequence and then later combined withthe rest of the gene to give a full length sequence.

In this example the amino acid sequence for the Pro-peptide part of theparent proteases given in SEQ ID NO: 28; SEQ ID NO: 33; SEQ ID NO: 37;SEQ ID NO: 41; SEQ ID NO: 43; and SEQ ID NO: 45 is encoded by a set ofoligonucleotides and the resulting shuffled gene fragments are combinedinto the context of the full length protease gene, which then consistsof DNA coding for the signal sequence, the (shuffled) Pro-peptide, andin this case the mature protein of 10R protease. Examples of shuffledPro-peptide sequences are shown in SEQ ID NO: 46 (0-2.19), SEQ ID NO: 47(G-2.73), SEQ ID NO: 48 (G-1.43), SEQ ID NO: 49 (G-2.6), SEQ ID NO: 50(G-2.5), SEQ ID NO: 51 (G-2.3), SEQ ID NO: 52 (G-1.4), and SEQ ID NO: 53(G-1.2).

The complete protease encoding genes were inserted into the genome of B.subtilis by homologous recombination as described above, and theproteases expressed in shakeflasks using a rich media. The fermentationwas carried out for 5 days at 30° C. and the supernatant isolated bycentrifugation prior to measuring the protease activity. As control a B.subtilis clone expressing the wild type protease 10R from Nocardiopsissp. NRRL 18262 from an identical construction protocol was fermentedunder the same conditions. The protease activity was calculated and ispresented in the table below relatively to the activity of the wild type10R protease. Clearly the heterologous pro-regions provide an advantageover the native pro-region of the 10R protease.

TABLE 21 Relative activity of 10R protease expressed with heterologousshuffled pro-peptides. Rel. acivity 10R 1.0 G-1.2 2.9 G-1.4 1.4 G-2.31.6 G-2.4 3.4 G-2.5 3.0 G-2.6 4.2 G-2.7 1.6

Example 16 In vivo Monogastric Performance of Tail-variant 10R-HV1

This example describes a dose/response study with the four amino acidtail variant HV1 of the 10R protease in the monogastric in vitro modelusing 10, 25, 50, and 100 mg EP/kg, and using 10R protease as benchmarkor control. The tail variant 10R-HV1 was constructed to have 4 aminoacids extra in the C-terminus: QSAP (SEQ ID NO: 5) as described above.

In vitro Conditions:

-   Substrate: 4 g SBM, 6 g maize (premixed)-   pH: 3.0 stomach step/6.8-7.0 intestine step-   HCl: 0.105 M for 1.5 hours (i.e. 30 min HCl-substrate premixing)-   Pepsin: 3000 U/g diet for 1 hour-   Pancreatin: 8 mg/g diet for 4 hours.-   Incubation: 40° C.-   Replica: 5    Enzymes:-   10R protease: FFE-2003-00047; batch PPA21400; 154 mg EP/g product.-   Freezedried 10R-HV1 FFE-2003-00077; 370 mg EP/g product    Solution A: 10R, 100 ma EP/ka Diet:-   100 mg EP/kg diet˜1 mg EP/flask    1 mg EP/mL-   (1 mg EP/mL*10 mL)/154 mg EP/g product=0.0649 g-   Prepare 10 mL: Disolve 0.0649 g enzyme in 10 mL NaAc buffer.    Solution C: 10R-HV1, 100 mg EP/kg Diet:-   100 mg EP/kg diet˜1 mg EP/flask    1 mg EP/mL-   (1 mg EP/mL*20 mL)/370 mg EP/g product=0.05405 g-   Prepare 20 mL: Disolve 0.0541 g enzyme in 20 mL NaAc buffer.    Solution D: 10R-HV1, 50 ma EP/kg Diet:-   50 mg EP/kg diet˜0.50 mg EP/flask via 1 ml=0.50 mg EP/ml-   Prepare 10 mL: Dilute C 2 times: 5 ml solution C+5 ml 125 mM    NaAc-buffer    Solution E: 10R-HV1, 25 ma EP/kg Diet:-   25 mg EP/kg diet˜0.25 mg EP/flask via 1 ml=0.25 mg EP/ml-   Prepare 12 mL: Dilute C 4 times: 3 ml solution C+9 ml 125 mM    NaAc-buffer    Solution F: I10R-HV1, 10 mg EP/ka Diet:-   25 mg EP/kg diet˜0.25 mg EP/flask via 1 ml=0.25 mg EP/ml-   Prepare 10 mL: Dilute C 10 times: 1 ml solution C+9 ml 125 mM    NaAc-buffer    Substrates:-   Premix (40% SBM/60% maize), FFS-2002-00121-   The 10 g sample contains 6 g maize and 4 g SBM giving a calculated    protein content of 23.48% of protein (˜2.35 g/flask).    Chemicals:-   4.005 M HCl, AT-1-00061/29-   4.007 M NaOH, AT-1-00002/36-   Pancreatin FFE-2002-00052, 8×USP-   Pepsin FFE-2003-00048, 471 U/mg    NaOH 0.39 M:-   Prepare 500 mL:-   48.97 mL 3.982 M NaOH, fill with milliQ to 500 mL.    HCl1 Solution 0.105 M-   Prepare 2000 mL:-   52.43 mL of 4.005 HCl, fill with milliQ up to 2000 mL    HCl2 (HCl/Pepsin) Solution: 0.105 M Containing 30000 U Pepsin/5 mL-   Prepare 250 mL:-   Take out approx. 150 mL from the HCl-solution, add 3.18 g pepsin and    fill up to 250 mL with the HCl solution.    125 mM NaAc-Buffer, pH 6.0:-   Prepared from a 2 M NaAc-buffer (KLu 04-07-2003/lab book 14169 p.    104)-   →12.5 mL 2 M NaAc-buffer, fill up to 200 mL with milliQ    Pancreatin Dissolved in 1 M NaHCO₃ Containing 8 mg Pancreatin/g    Diet:-   NaHCO₃-pancreatin is pre made, divided into portions and frozen.    Made 29-04-2003 and frozen, it is slowly thawed in refrigerator over    night. The stock preparation is described in lab book 14165 page    068.    Flow Scheme:

In the Premixing phase (t=0), 10 g substrate is mixed with 41 ml HCl1;then in the gastral phase (t=30 min) 5 ml HCl-2 (HCl/pepsin) +1 mlenzyme (or buffer) is added, and later (t=1 h) the pH is measured and 16ml water is added; and then in the intestinal phase (t=1 h 30 min) 7 ml0.39 NaOH is added, and later (t=2 h) 5 ml NaHCO3/pancreatin is addedand the pH is measured again twice (t=2 h 30 min & t=5 h 30 min); andfinally (t=6 h) 30 ml suspension is sampled for centrifugation. Eachsupernatant is immediately and carefully removed from the centrifugetube into glass tubes. The supernatants are split in two aliquots forfurther analysis. Results are shown in table 22.

TABLE 22 Treatment of samples in the monogastric in vitro model. EnzymeEnzyme dose/ Pepsin Pancreatin Sample Solution Enzyme pH kg diet: U/gdiet: mg/g diet: 1-5 1 ml Blank 3.0  0 mg EP 3000 8.0 Buffer  6-10 1 ml10R 3.0 100 mg EP  3000 8.0 Solution A (FFE-2003-00047) 11-15 1 ml10R-HV1 3.0 100 mg EP  3000 8.0 Solution C (PPA22873) 16-20 1 ml 10R-HV13.0 50 mg EP 3000 8.0 Solution D (PPA22873) 21-25 1 ml 10R-HV1 3.0 25 mgEP 3000 8.0 Solution E (PPA22873) 26-30 1 ml 10R-HV1 3.0 10 mg EP 30008.0 Solution F (PPA22873)Soluble and Digestible Protein:

The changes in the levels of soluble and digestible crude protein in thesoluble phase of the hydrolysates were determined using an ÄKTA HPLC(Superdex 30 peptide column). The results are shown in Table 23.

At a 10R-HV1 dose of 100 mg EP/kg diet, the level of Digestible proteinwas significantly increased by 9.8%, compared to Blank. The control 10Rshowed a relative improvement of 7.7%. With the lower enzymeconcentrations (50, 25, and 10 mg EP/kg diet) the relative improvementsof Digestible protein were 5.7%, 3.3% and 0.7%, respectively.

TABLE 23 HPLC results with 10R-HV1 and 10R showing the percentualchanges in digestible CP and soluble CP relative to blank. Differentletters on top of the bars indicate significant differences (1-ANOVA,Tukey, 95%). MoFi030043, day 2 Of total protein Relative to blank Enzyme[mg EP/kg] n % dig. CP SD % sol. CP SD % dig. CP CV % % sol. CP CV %Blank 11 54.8 1.2 83.9 1.6 100.0^(a) 2.3 100.0^(a) 1.9 10R HV1 [100] 560.2 0.8 88.3 1.1 109.8^(e) 1.3 105.3^(c) 1.3 10R HV1 [50] 5 58.0 0.686.9 0.8 105.7^(cd) 1.1 103.6^(bc) 0.9 10R HV1 [25] 5 56.7 0.8 86.3 1.0103.3^(bc) 1.4 102.9^(bc) 1.2 10R HV1 [10] 5 55.2 1.3 84.3 1.9100.7^(ab) 2.4 100.5^(ab) 2.2 10R [100] 5 59.1 0.8 87.1 1.9 107.7^(de)1.4 103.9^(bc) 2.2

The original 10R [100 mg EP/kg diet] improved the level of solubleprotein by about 4%. The effects of 10R-HV1 was slightly higher (5.3%relative increase) and significant. With a dose of 50 and 25 mg EP/kgdiet the relative improvements were 3.6% and 2.9%, respectively andsignificant. With 10 mg EP/kg diet the relative improvement was 0.5%.

Degree of Hydrolysis:

The degree of hydrolysis (DH) was determined using the OPA method.Results are shown in Table 24.

TABLE 24 Degree of Hydrolysis (DH) determined by the OPA method.Absolute as well as relative values are shown. Different lettersindicate significant difference (1-way ANOVA, Tukey 95%). Of totalprotein Relative to blank Enzyme [mgEP/kg] n % DH SD % DH % CV Blank 525.89 ^(a) 0.43 100.0 ^(a) 1.65 10R (FFE-2003-00047) 5 27.19 ^(bc) 0.67105.0 ^(bc) 2.46 [100] 10R _(HV1) [100] 5 27.89 ^(c) 0.36 107.7 ^(c)1.29 10R _(HV1) [50] 5 27.34 ^(bc) 0.57 105.6 ^(bc) 2.08 10R _(HV1) [25]5 26.42 ^(ab) 0.57 102.0 ^(ab) 2.16 10R _(HV1) [10] 5 25.52 ^(a) 0.9698.6 ^(a) 3.76

Tail-variant 10R-HV1 improved DH by 7.7%, compared to Blank. With thelower doses (50 and 25 mg EP/kg diet) of the protease the improvementsranged from 5.6-2.0%, respectively, in line with previous findings. Atthe lowest dose [10 mg EP/kg diet] no effect was seen. The original 10R[100 mg EP/kg diet] showed improvements of 5% relative to Blank.

The results of the HLPC ÄKTA analysis and the DH determinations clearlyshow that addition of the four amino acid (SEQ ID NO: 5) long tail to10R does not affect the performance of the 10R protease to anysignificant extent.

1. A secreted polypeptide having protease activity on soybean mealprotein and comprising at least three non-polar or uncharged polar aminoacids within the last four amino acids of the C-terminus of thepolypeptide and comprising an amino acid sequence: (a) that is at least95% identical to the amino acid sequence of the mature part of thepolypeptide shown in SEQ ID NO:37; SEQ ID NO:41; or SEQ ID NO:43; or (b)that is at least 95% identical to the amino acid sequence of the maturepart of the polypeptide encoded by the polynucleotide in SEQ ID NO:1;SEQ ID NO:2; SEQ ID NO:31; SEQ ID NO:36; or SEQ ID NO:40.
 2. Thepolypeptide of claim 1, which polypeptide comprises at least threenon-polar or uncharged polar amino acids within the last four aminoacids of the C-terminus of the polypeptide, and which polypeptide: (a)comprises an amino acid sequence which is at least 96% identical to theamino acid sequence of the mature part of the polypeptide shown in SEQID NO: 37; SEQ ID NO: 41; or SEQ ID NO: 43; or (b) comprises an aminoacid sequence which is at least 96% identical to the amino acid sequenceof the mature part of the polypeptide encoded by the polynucleotide inSEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 31; SEQ ID NO: 36; or SEQ ID NO:40.
 3. The polypeptide of claim 1, which polypeptide comprises at leastthree non-polar or uncharged polar amino acids within the last fouramino acids of the C-terminus of the polypeptide, and which polypeptide:(a) comprises an amino acid sequence which is at least 97% identical tothe amino acid sequence of the mature part of the polypeptide shown inSEQ ID NO: 37; SEQ ID NO: 41; or SEQ ID NO: 43; or (b) comprises anamino acid sequence which is at least 97% identical to the amino acidsequence of the mature part of the polypeptide encoded by thepolynucleotide in SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 31; SEQ ID NO:36; or SEQ ID NO:
 40. 4. The polypeptide of claim 1, which polypeptidecomprises at least three non-polar or uncharged polar amino acids withinthe last four amino acids of the C-terminus of the polypeptide, andwhich polypeptide: (a) comprises an amino acid sequence which is atleast 98% identical to the amino acid sequence of the mature part of thepolypeptide shown in SEQ ID NO: 37; SEQ ID NO: 41; or SEQ ID NO: 43; or(b) comprises an amino acid sequence which is at least 98% identical tothe amino acid sequence of the mature part of the polypeptide encoded bythe polynucleotide in SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 31; SEQ IDNO: 36; or SEQ ID NO:
 40. 5. The polypeptide of claim 1, wherein the oneor more non-polar or uncharged amino acid(s) is (are) added to theC-terminus of the polypeptide.
 6. The polypeptide of claim 5, whereinthe one or more added non-polar or uncharged amino acid(s) is one ormore of Q, S, V, A, or P.
 7. The polypeptide of claim 5, wherein the oneor more added amino acids are selected from the group consisting of SEQID NO:3, SEQ ID NO:5, QP, TL, TT, QL, TP, LP, TI, IQ, QP, PI, LT, TQ,IT, QQ, and PQ.
 8. The polypeptide of claim 1 which further comprises aheterologous pro-region from a different protease.
 9. The polypeptide ofclaim 1 which further comprises a heterologous secretion signal-peptidewhich is cleaved from the polypeptide when the polypeptide is secreted.10. The polypeptide of claim 9, wherein the heterologous secretionsignal peptide comprises an amino acid sequence having at least 70%sequence identity with the amino acid sequence encoded by the sequenceof polynucleotides 1 -81 of either SEQ ID NO: 2or SEQ ID NO:
 44. 11. Ananimal feed additive comprising at least one polypeptide as defined inclaim 1; and (a) at least one fat-soluble vitamin, and/or (b) at leastone water-soluble vitamin, and/or (c) at least one trace mineral.
 12. Ananimal feed composition having a crude protein content of 50 to 800 g/kgand comprising at least one polypeptide as defined in claim
 1. 13. Acomposition comprising at least one polypeptide as defined in claim 1,together with at least one other enzyme selected from amongst phytase;xylanase; galactanase; alpha-galactosidase; protease; phospholipase A1;phospholipase A2; lysophospholipase; phospholipase C; phospholipase D;and/or beta-glucanase.
 14. An isolated polynucleotide encoding apolypeptide as defined in claim
 1. 15. A recombinant expression vectoror polynucleotide construct comprising a polynucleotide as defined inclaim
 14. 16. An isolated recombinant host cell comprising apolynucleotide as defined in claim
 14. 17. The recombinant host cell ofclaim 16 which is a Bacillus cell.
 18. A transgenic plant, or plantpart, comprising a polynucleotide as defined in claim 14, or anexpression vector or polynucleotide construct as defined in claim 15.19. A method for producing a polypeptide comprising cultivating arecombinant host cell as defined in claim 16 to produce a supernatantcomprising the polypeptide.